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The Role of Cyclic AMP in Gonadal Steroidogenesis'

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					BIOLOGY                     OF     REPRODUCTION                               14,    30-53           (1976)



                                                 The         Role             of Cyclic               AMP                in Gonadal                  Steroidogenesis’

                                                                                                     JOHN                M. MARSH

                                                            Endocrine              Laboratory,      Department                                    of Biochemistry,
                                                                 The           University      of Miami,    School                                 of Medicine,
                                                                                                Miami,              Florida           33152




       There            has        been           a great          deal       of work            done,            by                                THE ROLE     OF CYCLIC     AMP IN
many             investigators,                           over          the         past        10         years                                      STEROIDOGENESIS        IN THE
                                                                                                                                                            CORPUS    LUTEUM
which            implicates                     cyclic        AMP             in several            gonadal
functions,                   but         a complete                    understanding                       of      its                The         main          function                ot        the        corpus            luteum                is
role       in          any             one          of       these            functions               is        still          the      synthesis                and          secretion                 of     progestins,                     and
lacking.             The           possible                mediatory                  role      of        cyclic               this     has        been         shown             in most               species               by in vitro
AMP             in      the        gonadotropic                         stimulation                  of     ster-              and      in vivo           investigations                     to be,            at least           to some
oidogenesis           in ovaries           and      testis                                     has    been                     extent,   controlled       by LH. In some      species,    such
studied       the      longest       and      probably                                         the    most                     as the    cow,      the human     and     the monkey,        LH
thoroughly,           but     even     this    action                                      is far from                         appears     to be the        only  controlling       hormone
being      completely          elucidated.          The                                    first   indica-                     while   in others,     such as the hamster       and the rat,
tion    that    cyclic     AMP    was involved    with    the                                                                  LH is probably      only    one                                of several                  controlling
control      of steroidogenesis      came    from the stud-                                                                    hormones    (Greep,      1971).                                 In a series                  of studies
ies of Haynes         and co-workers      who in a series   of                                                                 (reviewed              in the             report           by Savard     et al., 1965)
experiments                             (Haynes                   and             Berthet,                1957;                an in vitro             model             system           was developed      in which
Haynes                 et        aL,            1959;            Haynes              et al.,              1960)                the     effect         of        LH       on       progesterone                       synthesis                 was
showed                 that         ACTH                  increased   the                  endogenous                          determined                  in incubating                      slices           of corpora                    lutea
level  of              cyclic            AMP             in the adrenal                    cortex,    and                      obtained     from       cows                         in early         pregnancy.      The
that exogenous       cyclic    AMP mimicked         the effect                                                                 slices   of corpora       lutea                        tissue      were    incubated     in
of ACTH        on steroidogenesis.          Guided      by this                                                                Krebs-Ringer        bicarbonate                             buffer      in the presence
work,   other    investigators        began     to study        the                                                            of LH             or other            test      substances                    and        the      amounts
role of cyclic    AMP in the gonadotropic              stimula-                                                                of    progesterone       and 2013 hydroxypregn-4-en-3-
tion  of steroidogenesis           in the testis    (Hall     and                                                              one synthesized           were    measured.          It was found
Eik-Nes,                    1962)                and         the          ovary            (Marsh               and            that     LH produced        a marked        increase      in proges-
Savard,              1964a;                      Marsh             and         Savard,             1964b).                     tin synthesis      in these     incubating         slices   and this
From            these     early                   studies            on       gonadal            steroido-                     effect      was specific     for hormones            with   luteiniz-
genesis           the field                    has expanded                        to where      cyclic                        ing      hormone                      activity                (Mason                   and          Savard,
AMP             has been                     implicated     to                    some    extent        in                      1964a).
nearly           all        actions               of      luteinizing               hormone                (LH)                       After        the         initial        studies              on        the      effect            of      LH
and        some               actions               of      follicle           stimulating                      hor-           on    steroidogenesis                        in incubating          bovine    corpora
mone    (FSH)   in the ovary    and the testis.                                                                                lutea      slices      were                 completed,           the investigation
    I will attempt     to review    the studies                                                       on         the           logically         divided                 into    two     parts:      a) a study      of
role       of        this        cyclic            nucleotide                  in the         process              of          the    site          of LH action       on                               the           steroidogenic
steroidogenesis                            in       the      corpus               luteum            and          the           pathway;             and b) an investigation                                          of the mecha-
 testis,    since                  these           two       gonadal                tissues         seem           to          nism         of     action.             The       site        of     the        major            effect               of
reflect  contrasting                               situations.                I will also discuss                              LH    on the   steroidogenic             pathway      has   been
the possible       modes                             of action                of cyclic  AMP on                                shown   by numerous        investigators         to be between
the      steroidogenic                           pathway.                                                                      cholesterol                and        pregnenolone                       (lchii          et al.,          1963;
                                                                                                                               Hall      and        Koritz,               1964;          Koritz                and      Hall,            1965;
                                                                                                                               Hall      and        Young,               1968;          Armstrong                     et al.,           1970).
                                                                                                                               The       study            of     the        mechanism                     of       LH         action,            on
       ‘Dedicated                  to      Dr.     Karl       H. Slotta             (Emeritus             Profes-
sor of       Biochemistry     and Medicine      of the University                                                   of
                                                                                                                               the other    hand, has been largely  a study     of the
Miami)        on the occasion    of his eightieth     birthday.                                                                possible  role of cyclic AMP in this gonadotropic


                                                                                                                          30
                                                                                            CYCLIC                  AMP         AND        STEROIDOGENESIS                                                                                                          31


effect          and         this               is still      under                  investigation.                                           the       fact         that         human           corpora                lutea        of pregnancy
                                                                                                                                             have     a           much               smaller             capacity  to bind                                   1’    25
Effect          of Exogenous                                 Cyclic                 AMP
                                                                                                                                             labelled             human                LH or             hCG (Cole   et al.,                             1973;
       Marsh               and                 Savard      (1964a,                          1964b,                       1966b)              Rao         et    al.,  1975)                      and     to respond    to hCG   in
found               that             the          addition      of                       exogenous                         cyclic            terms          of adenylate                       cyclase,    cyclic  AMP accumu-
AMP            to        incubating                         slices             of      cow             corpora                lutea          lation            and           steroid             synthesis                    (LeMaire                  et        al.,
caused              a significant                           stimulation                          of progesterone                             1968;            Marsh           and        LeMaire,                1974a),              than        corpora
synthesis                  in terms                    of        mass,               [1-’        4c]         acetate             and         lutea of               the menstrual                       cycle.  If there     is a block
[73       H]         cholesterol                          incorporation.                                At         a concen-                 to hCG                 responsiveness                       in human     corpora      lutea
tration             of      0.02                  M the              magnitude                          of     the         cyclic            of pregnancy                         at      the      level         of       adenylate                cyclase
AMP            effect                was           about               equal                to     that            produced                  activation,                    as       these         experiments                         suggest,                   the
by       a saturating                            amount                   of        LH           and         the         pattern             exogenous                     cyclic          AMP          would             be able            to by-pass
of [1-’4C]       acetate      and                                    [7-3H]                   cholesterol                      utili-        that block.                    Hermier   et                 al.     (1972)              reported           that
zation   closely       resembled                                        that                produced                     by      LH.         exogenous                     cyclic   AMP                   also         stimulated                  proges-
Other            nucleotides                              such            as        3’-AMP,                   5’-AMP                  or     terone             synthesis                  in      a corpus                      luteum       of              the
ATP had                    no     effect                  in the system,      which                                       served             menstrual               cycle               and      the maximal                        response                 was
to establish                     a certain                  degree of specificity                                          to the            about            the          same          for      cyclic          AMP              as for         hCG.               It
action           of cyclic                       AMP.            The           effect             of LH              was        also         was        also reported        that                         theophylline,                      an         inhibi-
not       additive                        to       that          produced                          by         a maximal                      tor       of cyclic    nucleotide                             phosphodiesterase,                                 po-
amount             of            cyclic AMP,                                   indicating                   that           cyclic            tentiated                 the          effect         of          hCG            on     progesterone
AMP            might             be a mediator                                   of this               action             of LH.             synthesis    and      that                          imidazole,                     a stimulator                    of
The        concentration                                    of      cyclic  AMP                               needed     for                 this phosphodiesterase,                                   markedly                    reduced                   pro-
this      effect,      however,                                  far exceeded                                the endoge-                     gesterone                 synthesis                  in the                control              and     hCG
nous           level            in         tissues,              but            this         could             have            been          incubations.                   These                  results               are    in            complete
due      to an              inability                     of the               exogenous                      nucleotide                     accord        with    the    proposal         that      cyclic       AMP
to     readily               penetrate                       cells              or to its                    destruction                     mediates        the action     of LH and hCG on proges-
by cyclic      3 ,5’ nucleotide          phosphodiesterase.              In                                                                  terone      synthesis     in corpora      lutea.
this  regard,      the     dibutyryl         derivative        of cyclic                                                                          Dorrington         and    Kilpatrick        (1967)        reported
AMP,     which       has been        reported         to be resistant                                                                        that         5 mM                cyclic            AMP            slightly             increased                     the
to phosphodiesterase              degradation           (Posternak       et                                                                  synthesis        of                  progesterone         and                         20a     hydroxy-
al., 1962),     stimulated         progesterone            synthesis     in                                                                  pregn-4-en-3-one                            in incubating                             corpora       lutea
this system                          at     the much      smaller      concentration                                                         from           pseudopregnant                         rabbits              although              its effect
of 0.2     mM                             (Marsh,    1969).       Hall     and      Koritz                                                   on       interstitial                tissue         was           much            greater.           Hermier
(1965a,               b) carried                       out         a similar                      type         of experi-                    and         Jutisz              (1969)             showed                 that         3 mM                 cyclic
ment            using                     bovine   corpora       lutea                                        from     the                   AMP            stimulated     progesterone                                       synthesis         in incu-
estrous           cycle                    and showed      that,    like                                      LH, cyclic                     bating            slices  of luteinized                             rat          ovaries,        prepared
AMP            stimulated                          progesterone                              synthesis                    by       ac-       by        treatment                    of     immature                    rats        with       pregnant
celerating                  a step                 between                     cholesterol                         and        preg-          mare             serum              gonadotropin                          (PMSG)                and             hCG
nenolone.                                                                                                                                    according                 to the                procedure               of Parlow                    (1968).
    The               effect       of                     exogenous                        cyclic                  AMP      on               They      also             found                that    the          presence     of                 Ca2     in
progestin                synthesis                         has been                      assessed                  in luteal                 the        external                    medium                 was           required                 for         this
tissue          of         other                 species      as well.   LeMaire       et                                           al.      effect.
(1971)              reported                       a stimulation       of progesterone
synthesis       both    in terms    of mass and                                                              [i-’ 4C] ac-
                                                                                                                                             Endogenous                      Cyclic             AMP
etate     incorporation          in incubating                                                               slices  of a
human            corpus                          luteum             of ectopic     pregnancy.                                                      The results      with exogenous                                         cyclic            AMP were
The         effect      of                       cyclic            AMP    exceeded      that                                        of       compatible        with     the hypothesis                                         that          this nude-
human               chorionic                        gonadotropin                                (hCG),               but          the       otide     was a mediator         of the                                      action             of LH on
pattern              of     distribution                             of             4C among                       several            of     steroidogenesis                        in the         corpus              luteum,    but                   such a
the steroids        (progesterone,                                                17             hydroxyproges-                              consideration                        required           that            the addition                       of LII
terone,      androstenedione                                                    and               estradiol)                    pro-         would            bring     about                    an increase                     in the endoge-
duced   by the                             human    corpus    luteum                                        was           identi-            nous           concentration                          of cyclic                     AMP    in luteal
cal   in both                              the   hCG      and    the                                      cyclic               AMP           tissue.          Marsh              et al.         (1966)           showed               that        2 pg/mI
stimulated                      tissue                slices.             The               greater                effective-                of        LH       brought    about    a striking   increase    (as
ness      of        cyclic                 AMP              over          hCG            may            be related                    to     much             as 100 fold)    in the level of cyclic     AMP in
 32                                                                                                                      MARSH



incubating                        slices          of       bovine              corpora                lutea,           and        a      stimulation                             of         steroidogenesis                                     seems             to       be
this      response                     showed                  the       same            specificity                    for       about               10 ng/ml                    while                 the        amount                    of LU             needed
LH       as had                    been            previously                    demonstrated                           for       to        cause                the          first         significant                          increase                  in cyclic
the      stimulation                         of     progesterone                     synthesis                       (Say-        AMP       is                   about                 100          ng/ml      (Ling      and                                   Marsh,
aid      et        al.,          1965).             Furthermore,                        the         increase                 in   unpublished                               data).              It is possible       that the                                  inabili-
endogenous                          cyclic             AMP            preceded                  the          increase             ty        to         detect                  a change                        in         cyclic                AMP             at       the
in   progesterone                             synthesis      which    would                                     be      ex-       lower               concentration                                  of       LU           might             be       due         to     the
pected       if cyclic                         AMP      were    a mediator                                      of      the       fact         that              the          effective                   cyclic               AMP            was          confined
action          of         LH.         At         higher             concentrations                            (0.2       to      to         a         small                  compartment                                      of         the          cell.           One
2 g        of        LH/ml)                  there            was       a positive                  correlation                   component                             of       the            cell          which                 might             be       consid-
between                   the       increase                in endogenous                          cyclic            AMP          ered           as a compartment                                            is the             regulatory                   subunit
and        the stimulation                                  of progesterone       synthesis.                                      of        protein                     kinase.                   If         small              changes                 in        cyclic
At       0.02    pg LFI/ml,                                 however,      the results      were                                   AMP              did            occur                 when                low            levels    of                    LH were
less      clear.   Although                                  this concentration        of LH                                      used           they             might                 have              been            associated                       with this
usually      stimulated                             progesterone           synthesis,                                   ef-       regulatory                          subunit.                   We have                       recently               attempted
fects     on     cyclic                            AMP       levels      could        not                                be       to       detect                an interaction                             of cyclic                       AMP with                    this
regularly       detected.                            This      discrepancy          was                                not        subunit                   indirectly,                         by        measuring                         the changes                    in
considered                    to be a serious                              one in view                          of the            protein                    kinase                    activity.                     It          had            been            shown
variability                  of the incubating                              slice technique                        and            earlier               by            Goldstein                        and           Marsh                  (1972,                1973)
the      fact             that        methodology                          used           for         the        cyclic           and            Menon                       (1973)                       that    the     bovine                                corpus
AMP           measurements                              had         sufficient             experimental                           luteum                    had              a cyclic                       AMP-dependant                                      protein
error         to          obscure                  small             increases                in      the        cyclic           kinase.                Using                 the          procedure                           described                    by        Cor-
nucleotide.                                                                                                                       bin            et          al.            (1973),                       the   effect                          of         different
        Since             this       work              was         reported,             several              investi-            concentrations                                 of         LU            on protein                        kinase           activity
gators          (Beall               and          Sayers,             1972;        Catt            and         Dufau,             was     measured                                in            incubating                           slices    of               bovine
 1973;          Moyle                  and             Ramachandran,                               1973)              have        corpora      lutea.                             It        was              found                  that    there                was a
shown              that             low           concentrations                         of         tropic            hor-        significant                         increase                    in this                 kinase                activity               at 5
mones     can elicit     a stimulation                                        of steroidogene-                                    ng LU/mI,                             which                    is even                   slightly                 below                the
sis in other    tissues    without                                        a detectable        change                              minimum                             effective                     dose                  of         LI-I            which              will
in cyclic    AMP.      In the light                                       of these     reports       we                           stimulate                       steroidogenesis                                   (Ling              and Marsh,    un-
have          begun                  a reinvestigation                             of         the        dose            re-      published                       data).     It seems                              then,             in terms   of dose
sponse        relationships                                   of        LH   to   increases   in                                  response                       of the       bovine                           corpus                  luteum   to LH,
steroidogenesis             and                            cyclic        AMP    in the corpus                                     that            there                  is       good                    agreement                          between                     the
luteum.      In the older                                  study          of the dose                    response                 increase                      steroidogenesis,
                                                                                                                                                                 in                                                        the     increase     in
relationship       (Marsh                                    et        al.,    1966)                     we    only               protein                kinase    activity      and                                 probably       an increase
looked     for                   a change                  in cyclic            AMP         after   15 mm                         in     the          effective                  concentration                           of cyclic      AMP.
of incubation                        while                 progesterone                   synthesis    was                                 Puromycin,                            an        antibiotic                          which            inhibits               pro-
measured                     after            2        h      of      incubation.                      This            was        tein         synthesis,                       had             been          shown                  previously                      to be
because               we       had found    a maximum                                              response         in            capable                   of        inhibiting                       the         stimulatory                         effects             of
terms           of          cyclic    AMP    at 15 mm                                              when        large              both    LH    and                              cyclic                   AMP                  on steroidogenesis
amounts                     of     LH  were    used.  In                                           our      recent                (Savard    et al.,                              1965).                   This                 substance      had                        no
experiments                           (Ling                 and         Marsh,                  unpublished                       effect,               however,                           on          the          stimulation                         of        cyclic
data),             where               we           measured                    cyclic    AMP     by                          a   AMP            accumulation                                    by          LH       which                 indicated                  that
modification                           of         the    Gilman                    (1970)     method                              protein                   synthesis                           was            not              required               for              this
(Ling           et         al.,       1973),                 we        found             that           the           time        effect,               and              that              the            puromycin                             probably                  in-
course           of cyclic                        AMP      accumulation                             is nearly                     hibits               the               stimulation       of                               steroidogenesis                                at
linear          when   small                       amounts       of LH                          are used.     In                  some            step                after     the increase                               in cyclic       AMP.                        The
the      recent               dose     response       study,     therefore,      the                                              increased                       amount                     of           cyclic                AMP             produced                      in
amount                of        progesterone          synthesis        and  cyclic                                                these               corpora                    lutea               slices               has          been            found              by
AMP           were            both      measured        after   2 h of incuba-                                                    Goldstein                           and         Marsh                      (1973)                  to         be         localized
tion.         Our            preliminary         evidence       indicates      that                                               predominantly                                       in          the           cytosol                      fraction                  and
there         is still               a dissociation                        between                     the        mini-           about               25         percent                   of        this          cytosol                  cyclic             AMP             is
mum       effective                        doses           of LH which   will stimulate                                           bound                to        a macromolecule.                                          The         corpus                luteum,
steroidogenesis                             and            cyclic  AMP  accumulation.                                             as mentioned                                   before,                      also     contains        a                          cyclic
The minimal                          effective                dose of LH which        causes                                      AMP-dependent                                    protein                      kinase      (Goldstein                               and
                                                                               CYCLIC               AMP         AND           STEROIDOGENESIS                                                                                                            33

Marsh,             1972,           1973;              Menon,                1973)            and         most         of         although                  these          authors               did         report            a stimulation
this        enzyme                  is        also           located                 in      the         cytosl                  of cyclic                 AMP            accumulation         by LU in incubat-
fraction               (Goldstein                          and        Marsh,               1973).               It       is      ing slices                 of           isolated      corpora    lutea obtained
probable               that          this            protein                kinase           makes              up        a      from           6 or          18         day          pregnant                  rats,         they          did        not
major              portion               of          the          macromolecular                            cyclic               observe               an         increase                in      cyclic           AMP              in     corpora
AMP            binding                   material.                    Menon                  (1973)               also           lutea obtained                          from            a 20 day pregnant                                 rat or a
reported               that         some               preparations                        of      LH       could                rat which      had                       been           treated with   low                               doses  of
bring        about       a direct   stimulation                                        of his protein                            PMSG            (8         IU)         and      hCG              (12.5          lU).          No         measure-
kinase         preparation        when      added                                     to the in vitro                            ments           of          steroid             synthesis                     were           made           in        this
enzyme      assay.   This indicated                                           that LH might                          be          study,          so it is not                    possible             to correlate                     a lack            of
able    to produce         its effects                                        by a mechanism                                     response           of cyclic                     AMP              with   steroid                    synthesis.
independent        of cyclic   AMP.                                         We have confirmed                                    Ahren            et al. (1974),     in                            their     study    of corpora
this observation                        but have                      come     to               a different                      lutea          from    PMSG-treated                                   rats,    found    that    LU
interpretation.                     First of all,                     we found                  that only                 a      markedly                   increased                    the       content               of      cyclic           AMP
few batches   of LH                                  showed  this ability.                               Second-                 in 1 day old corpora     lutea.                                           In fact, the                   response
ly, when    we boiled                                  the LH preparation                                   it lost              was much    greater  than that                                             of isolated                   follicles.
it’s     ability             to     stimulate                      cyclic        AMP               accumula-                     As       the         corpora                 lutea            grew         older,            however,                  the
tion        and         progesterone                              synthesis             in       incubating                      response                  quickly              declined                until           at     Day         7 it was
slices        of       bovine        corpora                          lutea,          but         it did not                     very       small.            The         failure              of Mason                 et al. (1973)                    to
lose         its       ability                to           stimulate                 protein                kinase               detect          a response                         to     LU          in corpora                    Iutea         may
(Goldstein                   and         Marsh,                   unpublished                      data).            We          have          been          due         to the           chance             selection                   of ovaries
don’t  know      what    the stimulatory   agent      is, but                                                                    when           the         corpora             lutea           were           in this          decline.
we don’t    think     it is LH. We believe       therefore
                                                                                                                                Adenylate                    Cyclase             and           Cyclic
that  this direct                     effect of some                            LH preparations
                                                                                                                                 Nucleotide                   Ph osphodiesterase
is not part     of                   the mechanism                              of action     of                     LU
on steroidogenesis.                                                                                                                   The    increase     in endogenous         cyclic    AMP   in
   LU and hCG                                 also          brought             about              a marked                      luteal    tissue     could    theoretically         be brought
increase             in cyclic                 AMP               accumulation                      in human                      about    by a stimulation         of adenylate        cyclase  or
corpora              lutea         measured                    in terms              of mass (Marsh                              by       an     inhibition                    of        cyclic            nucleotide                    phospho-
et al.,             1966)           or in                   terms     of             [8-3Hjadenine                               diesterase       and     these       alternatives                                            were   investi-
incorporation                       into              cyclic            [8-3H]AMP                         (Marsh                 gated       in homogenates              of bovine                                           corpora    lutea
and        LeMaire,                1974a).                 The        effect          appeared               to be               by      assaying     the      effect        of LU                                           on adenylate
specific              for          human                     gonadotropin                           with             LH          cyclase               in         the         absence                 of        phosphodiesterase
activity             and showed                        about    the same                           sensitivity                   activity              (Marsh,                  1970a).                The         complete                   inhibi-
to hCG               as the effect                      on steroidogenesis                                in this                tion        of detectable                          phosphodiesterase                                   activity
tissue (Savard                      et al., 1965).   Corpora                                          lutea           of         was       accomplished                          by using a high                             concentration
the menstrual                      cycle   were much   more                                       responsive                     of theophylline                          (0.04            M) and a short                          incubation
to these             gonadotropins                               in terms             of        cyclic       AMP                 period.       Under                        these            conditions,                          0.1     pg             of
accumulation           or steroid                                  synthesis        than              corpora                    LU/mI                or      0.01    M NaF       significantly                                          increased
lutea      of      pregnancy                                      (Marsh         and                 LeMaire,                    adenylate                    cyclase   activity.         Epinephrine                                            at           a
1974a).       This     diminished                                    responsiveness                     of the                   concentration                           of 0.2           mM          also       produced                  a small,
corpus             luteum    of pregnancy                                 could              be due to a                         but statistically          significant        stimulation         of                                                  ade-
decrease              in the number       or                            quality              of hormone                          nylate    cyclase       activity,        but    LU inactivated                                                          by
receptor              sites,        as reported                        by Cole               et al. (1973)                       hydrogen        peroxide,         bovine     serum       albumin,                                                     pro-
and      Rao         et al. (1975).                                                                                              lactin,          ACTH                   and          glucagon                  were           inert         in        this
     Lamprecht                      et al.            (1973)      found                    that          isolated               respect.     The significance                                     of the effect                   of epineph-
corpora        lutea                  from               pseudopregnant                                  rats     or            tine     is uncertain      since                                 it was only                      assessed    at
luteinized               rat       ovaries                 also       responded                    in vitro           to         one        concentration                         and no thorough                                  study   has
10 pg         of       LH/ml              with              an increase                   in cyclic             AMP              been         carried     out                    on its effect  on                              cyclic   AMP
accumulation,           but    the    response       of isolated                                                                 accumulation                           or progesterone                           synthesis                 in incu-
Graafian      follicles     was more       striking.   Mason     et                                                              bated           slices             of        corpora                 lutea.            Fontaine                  et     al.
al. (1973)      also found       that LH was less effective                                                                      (1970)           have             observed                    a stimulation                     of epineph-
in       stimulating                     cyclic                  AMP           accumulation                           in         tine  of adenylate         cyclase      in homogenates                                                                   of
luteinized               rat ovaries    or ovaries                                    from           pregnant                    whole   ovaries    obtained        from   goldfish,    and                                                            this
rats than               ovaries    from   immature                                       rats.         In fact,                  effect          also             required                an       elevated                  concentration
 34                                                                                                                      MARSH


 of     the         catecholamine                            (0.05               mM).             In testicular                   ATPase                 made      it difficult                            to study.    The largest
 tissue,             epinephrine                        also               has         been           found              to       amount                 of activity       was                         associated    with a 700                                 x
stimulate                  adenylate        cyclase      (Murad                                   et al.,     1969;              g pellet,   indicating                                   that           this       enzyme      system     in
Pulsinelli                 and     Eik-Nes,        1970)      and                                 cyclic       AMP               rat corpora      lutea                                might            also       be associated       with
accumulation                            (Kuehl              et al., 1970a).    This cate-                                         the       plasma                      membrane.                   No          studies              were          carried
cholamine                        also       has            been    found    to stimulate                                          out       on          the          effect            of      LH        or other                  hormones                  on
testosterone                        secretion                   in         perfused               testes         (Eik-           this       enzyme                      system.
Nes,          1971),     but it is unknown                                             at this         time        if it
plays          a physiological     role.
     The              effect  of   LH    on   phosphodiesterase                                                                  Possible                    Role          of Prostaglandins                                in the
activity              also was evaluated    in homogenates                                                               of      Action                 of LH              on Adenylate                          Cyclase

bovine      corpora                       lutea:    no change      in activity       was                                                 Prostaglandins                                 were            implicated                      as      possible
observed       under                      a variety    of experimental          condi-                                           mediators                         of      LU action       when                         they     were shown
tions      (Marsh,                         1970a).      These      data     indicate,                                            to stimulate:                             1) progesterone                             synthesis      in incu-
therefore,       that                     LH brings      about    the in’rease         in                                        bating            slices                  of      bovine               corpora        lutea                   (Speroff
endogenous                        cyclic          AMP            in bovine                   corpora             lutea           and             Ramwell,                          1970);                2)     adenylate                        cyclase
by       a stimulation                         of      the           adenylate                    cyclase          and           activity                in homogenates                                 of bovine                corpora              lutea
not          by      an         inhibition                 of        the         phosphodiesterase.                              (Marsh,                     1970b);                   and        3)      the           incorporation                         of
Phosphodiesterase                                    also            was           demonstrated                          in       14       CI adenine                           into           cyclic            AMP               in        incubated
bovine,    human       and                            rat       corpora                 lutea         by Stans-                  whole             ovaries                     obtained             from               rats (Kuehl                   et al.,
field   et al. (1971),                               but        although                  the        properties                  1970b).              Marsh                      (1970b,               1971)             tested the                effects
of this enzyme                               in bovine    tissue   were examined,                                                of        six           prostaglandins                                (PGE1,                 PGE2,                PGA2,
no attempt    was                            made    to assess   the effect  of LU.                                              PGB,,                  PGF,                     and         PGF2,)                    on      adenylate                   cy-
        The           subcellular                      localization                          of      adenylate                   clase           activity    of homogenates                                            of bovine    corpora
cyclase               in bovine     corpora                                  lutea      has    been                  as-         lutea            and     found    that     PGE,                                        and    PGE2      were
sessed              by homogenizing                                   and        fractionating                      the          about             equally                      potent     and more    effective     than
subcellular   components       of this tissue   by isoton-                                                                       PGA2               or             PGB1.             PGF2a     and  PGFIa        did not
ic and hypotonic       methods    (Sidhu     et al., 1975).                                                                      produce              significant                     stimulatory                           effects.
The          fractions                  were           assayed                    for adenylate       cy-                             An           additivity                      experiment                      was          carried            out       to
clase          activity                 and          standard                      marker    enzymes.                            test  the possible                                mediatory                  role of prostagland-
Only          5’ nucleotidase                              paralleled                  the        distribution                   ins in the action                                 of LH on                 adenylate    cyclase.                            In
of    adenylate        cyclase                                   in both                     isotonic              and           each experiment                                   an aliquot                 of a homogenate                                of
hypotonic       procedures,                                     indicating                   that     the         ade-           a bovine                     corpus               luteum                was           incubated                   with         a
nylate    cyclase                   of bovine corpora     lutea                                     is primari-                  maximally        stimulatory                                       concentration                           of PGE2
ly localized                      on the plasma       membrane.                                          Menon                   (100     pg/mI).       Another                                      aliquot      was                     incubated
and      Kiburz      (1974)     have    also prepared       a mem-                                                               with            this
                                                                                                                                                    concentration                                  of PGE2      plus    LU (10
brane      fraction       from   bovine     corpora   lutea   which                                                              pg/mI).             third
                                                                                                                                                        A      aliquot                            was incubated       with LU,
contains        adenylate      cyclase,      [1251] hCG binding                                                                  and        a fourth       aliquot                              was incubated       alone  as a
activity              and    the   plasma        membrane           marker                                                       control.                    If      LH transmitted                              its        effect            on luteal
enzyme               Na-K-ATPase,       indicating       that     the hor-                                                       adenylate                          cyclase   solely                           via          PGE2,               then    it
mone              receptor     and   the adenylate           cyclase      are                                                    follows   that  a homogenate          of a corpus      luteum
both         localized                  on     the     plasma                    membrane.                                       which   was responding       maximally       to a saturating
        LH          has         also          been     shown    to stimulate                                       the           level of PGE2     should    show      no further    response
adenylate                   cyclase             activity     of corpora      lutea                                   of          when    LH was added.          It was found         that      LU
other   species,      such      as the rabbit     (Anderson          et                                                          produced                         a clear-cut  additive     effect                                      above     that
al., 1970)       and the pig (Anderson            et al., 1974).                                                                 produced                         by a maximally        stimulatory                                         concen-
In the latter        investigation,        it was found          that                                                            tration                of   PGE2,                     indicating            that               the           effect      of
there  was a decline          in adenylate     cyclase    activity                                                               LH        was            probably                       not        transmitted                         via       prosta-
and    LU                 responsiveness            which                              correlated      with                      glandins                (Marsh,                  1971).
the onset                    of luteal        regression.                                Stansfield     and                             Other                     investigators                     using              a similar                 experi-
co-workers                     have      carried        out                            studies      on the                       mental                  format                   have            reported                that                LU          and
adenylate              cyclase                 in rat corpora                           lutea   (summa-                          prostaglandins                                were          additive             in their               effects           on
rized     in          a review                   by Stansfield                           et al., 1972).                          adenylate         cyclase                             and      cyclic       AMP accumulation
Adenylate                       cyclase              was         detectable                       in homoge-                     in      a variety       of                        tissues              (Kuehl   et al., 1970b;
nates          of         rat      corpora                  lutea,               but         a very         active               Jonsson                     et         al.,       1972;               Kolena                and         Channing,
                                                                              CYCLIC                  AMP          AND          STEROIDOGENESIS                                                                                                           35


1972;           Lamprecht                      et al.,           1973).                                                            mouse          ovaries             (Kuehl               et al.,             1973).
        An          opposite              conclusion                       was           reached,                  how-                    Another                 approach                        to           evaluating                      prosta-
ever,          by      another                 group             of investigators                          (Kuehl                  glandins             as possible                      mediators                   of LH           action             has
et      aL,         1970b)              who            evaluated                   the           effect            of       a      been          the          use        of inhibitors                             of      prostaglandin
prostaglandin                          antagonist,                        7-oxa-1           3-prostynoic                           synthesis,                 such         as indomethacin,                                   aspirin                  and
acid, on              the stimulation    by LU and                                          prostagland-                           flufenamic                  acid.           These               compounds                        have              been
ins of              [1 4C] cyclic     AMP accumulation                                             in incu-                        reported              to        be ineffective                          in inhibiting                    the         LH
bated  rat ovaries.       They                                found    that   this antago-                                         stimulation               of    cyclic        AMP accumulation            in
nist not only       blocked                                  the effect     of PGE1      and                                       incubating              mouse      or rat ovaries     although       they
PGE2,       but                  also           competitively                             inhibited                 the            produced               a marked        inhibition  of prostaglandin
stimulation                    of the           accumulation                         of      [1 4C]            cyclic              synthesis             (Kuehl     et al., 1973;    Zoretal.,       1973).
AMP            produced                 by       LU.             They         concluded,                       there-              Indomethacin                          has      also                been            reported                   to       be
fore,  that    a prostaglandin                                           receptor                functioned                        ineffective                  in        inhibiting                     the            stimulation                       of
as a necessary      intermediate                                          in the          action              of    LU.            ovarian            steroidogenesis                            by        LH      when            this         prosta-
This      conclusion                         should    be viewed                                  with    some                     glandin             synthesis               inhibitor                    was         administered                         in
reservation,         since                    the same    authors                               indicated      in                  vivo         (Grinwich                    et          al.,         1972,             Tsafriri                et      al.,
a subsequent                          paper            (Kuehl               et      al.,         1972)              that           1972a).
7-oxa-1             3-prostynoic                      acid         was        not a completely                                             In    summary,                    four               general              approaches                       have
specific               antagonist,                    but          also         inhibited   cyclic                                 been         used          to     study               the       possibility                    that          prosta-
AMP-dependent                             protein       kinase.       In                        this       regard,                 glandins             mediate                the         action              of LH on adenylate
Marsh    and               LeMaire              (1974b)       carried                            out       experi-                 cyclase:             additivity                       studies,                studies of prosta-
ments  on the effect      of 7-oxa-1                                          3-prostynoic                          acid           glandin              antagonists,                        studies               of       prostaglandin
on [3 UI adenine     incorporation                                              into cyclic                        AMP             synthesis,             and     studies      of                          inhibitors                of         prosta-
in incubated      slices    of bovine      corpora                                                    lutea,     and               glandin             synthesis.        Although                                these             approaches
although     they     could    demonstrate         an                                                 inhibition                   have         not           yielded                a      definitive                   answer                 to      the
of the stimulatory                               effect  of LH                       with          very large                      problem,      the                 majority                of the              data appears                      to be
concentrations                          of        the antagonist,                               they    could                      in opposition                      to an                essential                role  for                   prosta-
not      show           that          it was          of the             competitive                     type.                     glandins              in        the         action                 of         LU        on            adenylate
     Channing                 (1972)      also                      found     that   7-oxa-13-                                     cyclase.
prostynoic                 acid inhibited                          the action      of PGE2     or
LU      on             luteinization                      in cultures                        of monkey                                              THE ROLE OR CYCLIC  AMP IN
granulosa                cells.     The              interpretation                        of this effect                                        STEROIDOGENESIS   IN THE TESTIS
was           complicated                       by         the       fact          that           when              this                  It    is generally                   recognized                        that       the          synthesis
inhibitor              was mixed      with prostaglandin                                                or LU it                   of androgens     in the testis        is under     the control
produced               severe  cellular    necrosis.                                                                               of LU (also     called   interstitial       cell stimulating
     A mediatory                          role     for prostaglandins         in the                                               hormone,     ICSU),    and       it was        reasonable      to
action   of      LH                       on     adenylate        cyclase     would                                                suspect         from    the work                              that had gone on in the
suggest    an     LH                       stimulation         of prostaglandin                                                    adrenal          cortex     (Haynes                              et al., 1960)     and   the
synthesis                 or   release.                          Chaslow         and                      Pharriss                 corpus         luteum      (Marsh                            and Savard,     1964b)    that
(1972)                reported        that                        the    injection                         of     LU               cyclic        AMP               was       involved                     in     this      action               of      the
antiserum                 into          rats         suppressed                    the      synthesis                   of         gonadotropin.                       The           investigation                        of       the       role         of
prostaglandin-like                          compounds                        from                [3    H] arach-                   cyclic         AMP     in steroidogenesis                                              in the    testis
idonic      acid               in      incubations                        of ovarian                   homoge-                     followed          a very similar      pattern                                        to the studies
nates,          and           that       LH          injected               into          the         animal            or         carried         out  in the     ovary.      It                                     began    with     the
added           to      the          incubation                   medium                  overcame                   this          study         of      the         effects              of       exogenous                      cyclic             AMP
suppression.                     Other               studies               also          indicated                  that           on steroidogenesis,                            progressed     to the assessment
LU     can              stimulate                 prostaglandin                             synthesis    in                        of adenylate      cyclase                         and finally    to the measure-
slices   of             monkey                  ovaries      (Wilks                        et al., 1972)                           ment          of       endogenous                             cyclic              AMP.                This          last
and incubated                          rabbit             Graafian       follicles     (Marsh                                      phase  of the                   work          has recently                         uncovered     some
et al., 1974).                       In the           latter     report,      however,      this                                   new problems                      for        investigators                        in this field,   and
stimulation                    appears                to         occur            beyond                the         ade-           these         problems                  will           be        discussed                  later            in     this
nylate            cyclase      step,                  because                it could                   be mim-                    section.
icked            by      exogenous                       cyclic                AMP.                   Dibutyryl
                                                                                                                                   Effects        of      Exogenous                        Cyclic              AMP
cyclic          AMP            has       also         been          reported                    to stimulate
prostaglandin                          synthesis                    in       incubated                         intact                     In     the       early           studies,                     whole            testis            prepara-
36                                                                                                                        MARSH


tions       were           used,             and         Sandier                   and        Hall            (1966)              dog         was        not             effective                   in stimulating                          testicular
were        the       first            to         show           that           exogenous                      cyclic             steroidogenesis,                               but          the          injection                 of     the         cyclic
AMP         could              stimulate               testosterone                           production.                         nucleotide                   directly       into the testis                                     in vivo           caused
They         used              teased              rat testis       and                      observed                   an        a considerable                     increase      in steroid                                    secretion.
increase             in          both              the           mass               of       testosterone                                  Leydig             cells          are            generally                    considered                     to       be
synthesized                    and          the conversion                          of [7a-3U]     cho-                           the       main         site of testosterone                                    production                        (Hook-
lesterol            into              [3     U] testosterone.                          The     amount                             er,       1970)            and the     effect                                of exogenous                          cyclic
cyclic       AMP               used          (0.027             M) was               far in excess                       of       AMP             has        also          been               studied               in        isolated             Leydig
physiological                levels,    but the effect     was specific,                                                          cell tumor        preparations.          Shin                                           (1967)             and Shin
since    ATP,              ADP and AMP had no effect              at this                                                         and   Sato      (1971)        demonstrated                                                   that          a mono-
concentration.                   Connell      and    Eik-Nes     (1968)                                                           layer   culture        of Leydig          cells                                           derived            from   a
confirmed                  this     effect      of                      cyclic    AMP      on                          the        mouse             interstitial                       cell         tumor,               which             was          insen-
synthesis             of       mass      amounts                           of testosterone                               in       sitive          to gonadotropins,                                       would            respond                to very
slices      of rabbit                  testis.           Their            report             corrected                  an        low         levels            of          exogenous                          cyclic                AMP           with             a
impression                 brought                  about               by         a previous                  study              significant                 increase                  in progestin                          synthesis.                Near-
(UalI       and          Eik-Nes,                  1962)               that        rabbit            testicular                   ly all          of the studies                            carried             out        with            exogenous
tissue           would               not          respond                 to        cyclic            AMP,               as       cyclic           AMP or even                              dibutyryl                    cyclic            AMP have
judged     by the                    incorporation                        of         [1-’4C]              acetate                 used            amounts                   of         these              nucleotides                      above              the
into   testosterone.                       Connell                      and          Eik-Nes              (1968)                  physiological                          level              of        cyclic             AMP,              but          these
also       assessed            the     effect                       of theophyllmne           and                                 workers              found   that    this preparation       of Leydig
found         that         it stimulated                          testosterone      synthesis                                     tumor            cells could      be stimulated       by as little as 5
at low           concentrations                          (107                M),         indicating                that           x      106          M cyclic    AMP.     This                                       is well within    the
there was a cyclic                            nucleotide       phosphodiesterase                                                  range             of  the    endogenous                                             concentration       of
and an adenylate                             cyclase     system     in testis    tissue.                                          cyclic           AMP     in normal      Leydig                                        cells (Rommerts
Other        investigators                        (Dufau                et     al., 1971,                     1972a,              et       al.,         1973;               Dorrington                            and            Fritz,             1974).
1972b;          Catt       et                al.,     1972;                   Rommerts                        et al.,             Although                     this              concentration                                  of         exogenous
1972)         have             demonstrated                            that        dibutyryl                   cyclic             cyclic            AMP                  was            quite                low,             its         continuous
AMP        is        capable     of accelerating           testosterone                                                           presence               was         required              to sustain    the stimulation
production               in whole    testis     preparations.            This                                                     of     steroidogenesis.                               The same      phenomenon                                             was
analogue             is about   50 times       more     effective       than                                                      observed               by          Moyle                  et      al.     (1971)                using           another
cyclic        AMP                (Catt     et            aL,           1972),          and    its                  time           mouse              Leydig                 cell             tumor                preparation,                      which
course         of              stimulation                       of           steroidogenesis                                is   was sensitive                      to both    LH and cyclic    AMP.                                                    This
almost            identical                   to         that            produced                        by        hCG            preparation,                       however,     required   the usual                                                   high
(Dufau           et al.,         1972a).                                                                                          concentrations                        of cyclic     AMP  (2 mM to                                                      0.02
     The          infusion                   of     cyclic               AMP               into          the        dog           M) to stimulate                                      steroidogenesis                                (Moyle                 and
testis via the spermatic        artery                                          in vivo also                       gave           Armstrong,     1970;                                   Moyle        et                  a!.,         1971).                The
rise to an increased     production                                              and secretion                        of          stimulatory                   effect                 of      exogenous                    cyclic   AMP                       on
testosterone          (Eik-Nes,        1967,    1969).    Again     large                                                         steroidogenesis                           has             thus    been                  well established,
doses      (150-300          pg/mi)      of this nucleotide         were                                                          but     the           mechanism                             by          which            it works                 is still
required,         but    the    effect       was specific       for    the                                                        uncertain.
cyclic       nucleotide.                      Van           der Molen                       and Eik-Nes
                                                                                                                                  Adenylate                  Cyclase
(1971)        using      the                same          technique                       demonstrated
that the           effect     of              cyclic         AMP                occurred                  at steps                       Another     approach    to the investigation          of the
in the             steroidogenic                           pathway                   prior                 to   the               role     of cyclic    AMP in testicular      steroidogenesis
formation                 of         pregnenolone.                             These              data         taken              was      the study      of the adenylate      cyclase      system
together            with          the         stimulation                      of the             conversion                      in this           tissue            and         its response                        to gonadotropins.
of       [7o-3      U] cholesterol                              into           3     U] testosterone                              Murad    et al. (1969)                                         found      that    both LU and
by cyclic             AMP     (Sandier   and  Hall, 1966)   indi-                                                                 FSH would      stimulate                                        adenylate      cyclase activity
cate that             the site of cyclic     AMP action   in the                                                                  in whole    homogenates                                            of dog       and rat testes.
testis   is somewhere                                   between     cholesterol                                    and            The         effect             of          FSH                 appears                 to          be     due          to         a
pregnenolone,      just                            as      it seems     to be.                                in    the           stimulation                       of         an           adenylate                      cyclase                 in         the
corpus        luteum                 (Uall and                  Koritz,              1965b)               and          the        seminiferous                        tubules                  and            the          LU effect                     to a
adrenal          cortex               (Karaboyas                      and                Koritz,              1965).              stimulation                    of      this               enzyme                  in     interstitial                   cells
Mieno         et al.           (1973)              found               that         the       injection                 of        (Cooke                et       al.,            1972;                Dorrington                          and           Fritz,
cyclic       AMP           into             the     systemic                   circulation                     of the             1974;            Braun              and              Sepsenwol,                        1974).             NaF              also
                                                                             CYCLIC                 AMP       AND        STEROIDOGENESIS                                                                                                                 37


produced                 a marked      stimulation,                                 and high con-                          nated         c and                f (Monn                     et al.,             1972;            Christensen
centrations                of epinephrine           and                             ACTU    caused                         and        Desautel,                     1973).                The          f form                 is a low                 Km
small         increases                in the           activity            of this            enzyme             in       type        (2.5            x      10-6            M)          and         is found                     only       in the
whole    homogenates          of dog testis.   Glucagon        had                                                         seminiferous                       tubules.                The             c form                is a high                  Km
no effect.      It is difficult     to suggest    any physio-                                                              type       (6.5         x        10-5       M) and                   is presumably                             found           in
logical    explanation          for  the small     stimulation                                                             the       other     parts     of                         the         testis.             The             amount                of
produced                 by          ACTU               since           it is not               known           to         total       phosphodiesterase                                       in the              testis            increases             5
have      any           effect          on        testicular               steroidogenesis                      or         fold        from  Day                     20       to Day                    50 of life in the                                rat
spermatogenesis,         and there     is disagreement            on                                                       and       then remains                          essentially                   constant  to Day                                 80
whether       or not it has any effect        on cyclic       AMP                                                          (Monn             et        al.,         1972).                This             increase                 in      enzyme
accumulation         in whole     cell testicular        prepara-                                                          production                       is very            similar                 to      that           of         adenylate
tions    (Kuehl    et al., 1970a;     Cooke       et al., 1972).                                                           cyclase           in this              period             of the                rat’s       life         (Uollinger,
The           epinephrine                        effect,             however,                   has       been             1970).            LU             has      no        acute                 effect           on           phosphodi-
observed       by                 other             groups    (Pulsinelli                           and     Eik-           esterase            (Kuehi                 et al.,              1970a)               and           its        effect          on
Nes,     1970;                    Kuehl               et al.,    1970a)                             and      this          steroidogenesis                            is        probably                       transmitted                         only
catecholamine                          has       also         been         found               to     cause          a     through            adenylate                       cyclase.
small    increase      in testosterone      secretion                                                 when   it
is infused        into   the     dog testis    in vivo                                                via the
                                                                                                                           Measurement                         and         Endogenous
spermatic                   artery                (Eik-Nes,                   1971).                  Eik-Nes
                                                                                                                           Cyclic   AMP
(1971)            suggested                    that this effect    may                                involve
nerve          fibers     and                the regulation     of the                               vascular                      Studies             carried                out         on         the       changes                    in endog-
system            of          the       testis.            This       might               explain    the                   enous   cyclic                      AMP         in the                     testis          have,     however,
stimulation                     of      adenylate                  cyclase               by epineph-                       not   completely                             supported                         the          proposal        that
rime,          since             elements      of nerve       and     vascular                                             cyclic            AMP                  mediates                     the          effect                 of      gonado-
tissues            must            be included       in homogenates             of                                         tropins            on           steroidogenesis.                            When             relatively                     high
whole           testes.          A similar    effect   of epinephrine         has                                          concentrations                       of gonadotropins                                       (greater                     than
been          reported                 in homogenates                         of        corpora           lutea            10 mg/mI       of                  LU or 1 ng/ml      of                                 hCG)     were                   used
(Marsh,      1970a),                          and       whole      ovaries                          obtained               they         have               repeatedly                      been               found                 to      cause              a
from    fish (Fontaine                              et al., 1970).                                                         marked              increase                    of endogenous                                cyclic              AMP in
     Uollinger        (1970)     and  Eik-Nes                                                  (1971)           re-        whole             testis     in                  vitro   (Kuehl                             et al.,               1970a;
ported       that    most    of the adenylate                                                   cyclase          of        Rommerts     et al.,                            1972;            Catt            et al., 1972;    Dufau
the testis        was found     in the “nuclear”                                                    and        the         et aL, 1973;    Catt                             and            Dufau,              1973;   Braun    and
“mitochondrial”                               fractions,               but         these     fractions                     Spensenwol,                        1974).          A similar     effect                               is apparent
were     identified                          only        by          their           sedimentation                         in Leydig                   cell         preparations        (Cooke                                 et al., 1972;
characteristics.                        No        enzyme              marker               or electron                     Cooke          et al., 1973;    Moyle   and                                                Ramachandran,
microscopic                          data          were            presented.                 Pulsinelli                   1973;          Rommerts      et al., 1973;                                                 Dorrington                        and
(1972)            assessed        the   correlation                                     of adenylate                       Fritz,         1974).                    This          stimulation                          appears                    to      be
cyclase           with     marker     enzymes                                for        plasma   mem-                      quite         specific                    in         that      prolactin,                        ACTU                        and
branes            (Na-K                 dependent                    ATPase)                   and        mito-            growth   hormone        do not                                                  increase                  the       cyclic
chondria        (succinic                       dehydrogenase)                             and found                       AMP levels    in either   type                                             of      testicular                   prepara-
the     cyclase       to                     be primarily                         associated      with                     tion        (Cooke                  et      al.,         1972).                  FSU         was               found           to
plasma       membranes.                           Sulimovici                        and Lunenfeld                          increase      cyclic   AMP     in                                         whole       testis    prepara-
(1973)            also       described      a hormonally                                  responsive                       tions,   but,     as mentioned                                            before,        it appears      to
adenylate                 cyclase      in a testicular                              mitochondrial                          act only on the seminiferous                                                  tubules        and not on
preparation                      which            was         relatively                free        of    endo-            the Leydig                       cells (Cooke       et aL, 1972;    Braun     and
plasmic      reticulum                         (Sulimovici       et al.,                       1973),       but            Sepsenwol,                        1974;   Dorrington       and Fritz,     1974).
the     degree        of                     possible      contamination                                   with            In regard               to         the       time              course              of the               hCG        effect,
plasma             membranes                            was          not           established.                     It     the rise in endogenous       cyclic                                                AMP appears                           after
seems,           therefore,                      that        most            of     the         adenylate                  about    10 mm     of incubation                                                    with hCG,                           while
cyclase    is located in plasma                                               membranes,                    but            the increase       in testosterone      production  was not
there   may also be an association                                              of this               enzyme               apparent     until    after     30 to 60 mm of incubation
with    mitochondria                                and         other         subcellular                  com-            (Rommerts      et al.,      1972;    1973).     This      is very
ponents      of the                    testis.                                                                             similar   to the    kinetics      of the    stimulation          of
        The       rat         testis         also         contains                two      isoenzymes                      cyclic    AMP    accumulation           and     progesterone
of      cyclic            nucleotide                       phosphodiesterase                              desig-            production                        by           LU             in          incubating                          slices            of
 38                                                                                                                           MARSH



corpora                  lutea           (Marsh                      et      al.,           1966),               and           is          The            fact    that                5 mM theophylline                                         by itself
compatible          with    the proposal                                                of a mediatory                                caused              a significant                  increase  in the                                    release     of
role     for    cyclic     AMP       in the                                              stimulation                          of      testosterone                  into         the           medium                     by      the        incubated
steroidogenesis          by gonadotropins.                                                                                            testicular               tissue,          and            potentiated                        the        effects             of
        When             careful                  comparisons                          of      the         dose           re-         low concentrations                                  of hCG,   indicated                                     to these
sponse              of    cyclic              AMP              accumulation                          and         testos-              authors   (Cart                    and              Dufau,  1973)       that                                  a small
terone         production                           have been                       carried             out,           how-           and        as yet undetectable                                       amount              of cyclic       AMP
ever,         a dissociation                            of these                      two            effects             has          was          probably     involved                                    in the               steroidogenic
been        uncovered.        In                         brief,   a hormonal                                stimula-                  response     to low hCG                                  levels.       This stimulation                                  of
tion       of steroidogenesis                                 can be elicited                              with low                   testosterone      production                                       by theophylline                                     was
concentrations                          of         LU          or     hCG            without               a detect-                  also observed       without                                     a detectable     increase                                in
able        change                in        the         endogenous                          level         of        cyclic            the        amount                 of          cyclic                 AMP             released                 to       the
AMP.          This            is a crucial                     discrepancy,                     since            one          of      medium.                It is           reasonable                          to expect                    that     this
the    criteria                    for             a         role           of     cyclic      AMP    in                              classical            inhibitor             of cyclic                       nucleotide                   phospho-
hormonal                      action               is         that           there      should     be a                               diesterase               acts    via cyclic                             AMP,   or cyclic    GMP,
demonstrable                        effect                    on the                concentration                             of      since      both             nucleotides                              are substrates      for this
this   cyclic                  nucleotide                       by the               smallest                  level          of      enzyme               (Goldberg                     et         al.,         1973).           Recently                   Po-
the hormone       capable      of                                     producing                  the physio-                          desta    and            Dufau       (1975)    reported    that there was
logical  response      (Robison                                           et al.,               1971).   Two                          a similar              dissociation        of the effects     of hCG  on
groups              of      investigators                            have     carried     out                        quite            testosterone        synthesis                                and             the    stimulation                            of
thorough                  studies       on                   this      problem        in the                        testis,           protein      kinase    in an                            isolated               Leydig      cell                 prepa-
and I will,                   therefore,                     review            their          work             in some                ration.             An           increase                      in          protein                kinase     was
detail.                                                                                                                               detectable                  at     100-250                           pg      hCG         and         reached     a
        Catt and                 Dufau      (1973)                         compared  the effects                                      maximum                     at       5        ng         hCG.                The         corresponding
of       various                 concentrations                               of hCG    (specific                                     dose-response                  range   for                            testosterone                      synthesis
activity            10,000   lU/mg)                                  on the release of testos-                                        was 5-100                   pg of hCG.
terone            and cyclic    AMP                                 into the medium    during                                                The          other          major                study               on       this         problem                  in
a 2 h incubation       of intact       decapsulated                                                                    rat            gonadal     tissue                  was             carried                  out     by            Moyle    and
testes. It was   found   that    testosterone                                                                   release               Ramachadran                       (1973).               They                 assessed              the effects
was        significantly                      increased       by                       hCG           concentra-                       of various             concentrations                                 of LH on testosterone
tions          as low                    as      0.1    ng/ml                          and           reached                   a      synthesis              and    cyclic                     AMP             accumulation      in                              an
maximum                       value           at         0.8          to       1.0          ng/ml.             On        the          isolated            Leydig             cell preparation    from      rat testis
other hand,  the release    of cyclic  AMP                                                                 began    at                and      a         mouse             Leydig     cell tumor     preparation.
1 ng hCG/ml     and reached    a maximum                                                                   at about                   The          testosterone                       was             measured                    after           2      h       of
100 ng/ml.  The cyclic    AMP was assessed                                                                   only at                  incubation                  and        the         cyclic                 AMP          after           10       mm         in
one        time          period              (2     h) and                  only        in the             medium                     both          the        medium                    and           the         tissue.           Again               there
in this           report,             but         thorough                   time           studies             from           1      was        a striking                difference                           between              the          amount
to      240          mm           had              been              carried            out           previously                      of LU required                            for       an increase                     in testosterone
(Catt          et      al., 1972;                        Dufau   et al.,    1973)       with                                          production    and                        that        required                    for a stimulation
1-500                ng hCG/ml                           and   no transient       rises     in                                        of cyclic       AMP     accumulation                                            in incubations       of
cyclic            AMP             were    detected.         It                              has also                 been             either    tissue.    The    amount                                         required      for a mini-
reported            that          the relative      changes                                  in cyclic               AMP              mal increase       in cyclic     AMP                                       was about       one order
appeared                 to      be         more              prominent                     in the             incuba-                of      magnitude                    (approximately                                    10         ng        LU/ml)
tion        medium                     than              in         the        testis           tissue               itself           above          that         required                    for          a minimal                    stimulation
(Dufau             et al., 1973).                            In addition,     in both   of the                                        of steroidogenesis                              (approximately                           1 ng LU/mI)
studies           just mentioned,                               an assessment        of cyclic                                        in the isolated                    rat           Leydig     cells.                   A time study    of
AMP           accumulation                              in      the         medium                  was         carried               the changes   in cyclic     AMP with these          rat Leydig
out using the                    incorporation                               of 14C-ademine                          into             cells  was carried      out     and    showed      that   cyclic
cyclic  AMP.                     An increase                              in 14C-adenine                          incor-              AMP accumulated          progressively        from    2.5 to 60
poration                 was       found                 to         parallel            the          changes                  in      mm,          but      again            this         study                 was       carried             out        only
the       mass           amounts                    of         cyclic          AMP             accumulated                            with  relatively  high concentrations         of                                                        LU         (500
when       20-500      ng of hCG/ml      were                                                  added             to the               ng and 5 pg LH/ml).
incubation        medium.    This   indicated                                                    that            newly                   The results   of another    very interesting                                                                 exper-
synthesized                     cyclic          AMP   comprised                                      a constant                       iment,         reported     in this paper,  indicated                                                    that the
proportion                    of the          cyclic AMP released.                                                                    increase          in cyclic   AMP, observed       with                                                 relatively
                                                                                   CYCLIC              AMP           AND           STEROIDOGENESIS                                                                                                 39


high      concentrations                              of LU,                 was      not       required                  for         work          would           seem          to       be     necessary               to determine
this      gonadotropic                        stimulation          of                        steroidogene-                            the         validity   of this suggestion,                                 but it does seem
sis.     It had      been                  shown        previously                             (Moudgal                      et       that         the large    increase  in cyclic                               AMP accumula-
al.,     1971;           Moyle                  et         al.,       1971)           that         LU            specifi-             tion      in the Leydig     cells of                              the      testis         caused           by
cally       binds             to        these              tumor               Leydig           cells,            stimu-              relatively      large   concentrations                                        of         LH     is        not
lating         testosterone                          production,                      and       that             a wash-              required             for      an increase                  in steroidogenesis.
ing      procedure                      can           not remove                        the bound                        LU                   Similar            dissociation               of cyclic             AMP           accumula-
nor       terminate                      the            enhanced                       steroidogenesis.                               tion    from               the effects     of ACTU        on corticoster-
The       bound                hormone                          could           be       removed,                    how-             oidogenesis                  (Beall    and   Sayers,    1972;    Moyle      et
ever,      by         treatment                       of        the         washed             cells         with           an        al., 1973;                Richardson       and Schulster,      1973)      and
antiserum                to       LH,             and   this   did                           terminate                   the          from           the         effects          of        TSU           on      thyroid               colloid
accelerated                    rate              of steroidogenesis.                                         On          the          droplet              formation                (Williams,                 1972)            have          been
other          hand,           it had                been             shown             that         exogenous                        reported.
cyclic          AMP     mimicked                                  the          effect            of the   hor-                                It seems,             then,         that          the     question               of the          role
mone           on steroidogenesis,                                      but       this         effect   by the                        of cyclic       AMP as a mediator              of steroidogenesis
cyclic          nucleotide                       could                 be      rapidly               terminated                       in testicular         tissue   is still unanswered.        There    are
by simply                washing    the tumor                                         cells. The inter-                               several       reasons        why      small   changes       in cyclic
pretation              of this data    was that                                       LU was retained                                 AMP concentration                                might           not be detected,                       even
by the cells even        after     repeated      washings,       and                                                                  if they  are      present,                           but           until there                   is     firm
 that the continued         presence        of the bound          LU                                                                  evidence              for a change                    in cyclic   AMP at the                             low
was required      for maintenance             of the steroido-                                                                        level    of          gonadotropin                      or another    messenger                              is
genic   response.    Since     the dose response           studies                                                                    discovered,                  this      question                 will      remain           in doubt.
just   described       (Moyle         and      Ramachandran,
1973)           raised             some               questions                    about             the         role        of
cyclic      AMP                    in      the   hormonal         stimulation                                                of                         POSSIBLE                 MODES        OF ACTION                        OF
                                                                                                                                                    CYCLIC            AMP         ON       STEROIDOGENESIS
steroidogenesis,                           a similar      washing       experiment
was       carried              out         by          these                authors             to         see       what                    If       we         believe            that          there           is      considerable
effect         this   procedure                             would               have on the endog-                                    evidence          in favor of a role for                                 cyclic      AMP in the
enous           level    of cyclic                           AMP.               It was found    that                                  action          of LH on steroidogenesis                                          in the corpus
although     the                    washing       procedure                             did not                    affect             luteum            and        possibly             the action       of high                      concen-
the steroidogenic                          response       to                          a relatively                    high            trations            of       LU on               Leydig     cells,    then                       we are
concentration                   of              LU (100                      ng/ml),           it did result                          confronted                with         the problem     of how this effect
in a rapid             cessation                  of cyclic                    AMP           accumulation                             might           be    carried           out. As mentioned     previously,
caused     by the                       LU. It was proposed                                        from      this                     the site of                action     of           LU       and cyclic              AMP           on the
data   that there                       might  be two types                                     of receptors                          steroidogenic                    pathway                   has been               shown            to be
for      LH:       one         which                  had          high          affinity              (since            the          between              cholesterol               and pregnenolone                                 (Ichii    et
effect could                   not be terminated                                      by       washing)    and                        al., 1963;             Hall and               Koritz,  1964,    1965b;                             Koritz
was involved                    with steroidogenesis,                                           and another                           and    Hall,             1965;    Hall and                        Young,     1968;                   Arm-
which      had            lower   affinity    (since                                      the effect                     was          strong    et          al., 1970).     There                       are, however,                   several
terminated                by washing)      and was                                       associated                     with          ways   by which                       cyclic  AMP      might    bring  about
the       major            increase                        in         cyclic          AMP              synthesis.                     the acceleration                       of this reaction      and these     are
Recently               Moyle     et al.                           (1975)      investigated                            the             shown    in Figure     1, and     are                                     listed  briefly     as
role of          the    carbohydrate                                 portion     of hCG                          on the               follows:   It could   bring   about                                       1) an increase      in
ability          of     this    hormone                                 to stimulate                             Leydig               the amount    of a cofactor     such                                     as NADPH;        2) an
cells      from      rat                testes     to synthesize                                testosterone                          increase             in     the concentration                          of the substrate,
and       accumulate                        cyclic    AMP.       They                             found      that                     cholesterol;                 3) an increase                       in the availability                        of
the removal                 of part of the                               carbohydrate                            of hCG               this        cholesterol               by      promoting                   its transport                  into
resulted    in             a marked    loss                               in the ability                           of the             the         mitochondrion,                       where            the      cholesterol                  side-
derivative               to         stimulate                         cyclic          AMP              accumula-                      chain          cleavage              system               is located;               4)     an     activa-
tion      but          a much                   smaller                  decrease               in         its      effec-            tion         or an increase    in the     synthesis                                      of       one        of
tiveness       to stimulate                                   steroidogenesis.                              Again             it      the         components      of the    cleavage                                      system;             5)     a
was      suggested      that                                there      were                  two           types             of       decrease              in      some          restraining                  influence               on      this
receptors                for            LU.    One                       which            mediated                     the            enzyme        system,     perhaps                                   by     enhancing       the
steroidogenesis                          response                        and            another                    which              transport      of an end-product                                      inhibitor,     such    as
mediated                 cyclic                 AMP                   accumulation.                          Further                  pregnenolone,         out of the                                mitochondrion.         There
 40                                                                                                                                  MARSH



                                         LH                                                                                                     tion        accelerated                      glycogen               breakdown,                    resulting
                                                                                                                                                in    the            formation                       of       increased                amounts                     of
                                                                                                                                                glucose              6-phosphate;                            3) the                metabolism                      of
                                                                                                                                                glucose             6-phosphate                       through               the      pentose              phos-
                     /-\
               AlP                                         CYCtIC            AMP                                                                phate  pathway                           produced                   an      increased               level          of
                                                                                                                                                NADPU,      which                          stimulated                     corticosteroid                        pro-
                                                                    i
                                                                                                                                                duction               by means                        of its role    as a required
                                                           I PROTEIN                I_#{149}.4.            PHOSPHORYLASE
           CHOLESTEROL                             .          KINASE                ]                                                           cofactor              in many                   of     the steps  of steroidogene-
               ESTER                                                 :
                                                                                                                                                sis,      including                the        cholesterol                 side-chain               cleavage
           (
           CHOLE5ROL
                     FREE
                                    ,,        0
                                                  / I        PRoTEIN

                                                            SYNTHESIS
                                                                                        I
                                                                                                 “S
                                                                                                       \        NADPH                           reaction.
                                                                                                                                                reaction
                                                                                                                                                                       The  enzyme
                                                                                                                                                                     has been   shown
                                                                                                                                                                                                              system
                                                                                                                                                                                                                to be
                                                                                                                                                                                                                                    for
                                                                                                                                                                                                                                  localized
                                                                                                                                                                                                                                            this          latter
                                                                                                                                                                                                                                                         in the
                                                   \                                                                                            mitochondria                       of         the         adrenal           cortex             cells       (Hal-

                                                                                                                           )
                            \_#
                                                                                                                       5
                                                                                                                                                kerston             et      al.,         1959,              1961)            and         the       NADPH
                E                   STEROL             .      *-        *-              PREGNENOLONE                        %
                                                  MITOCHONDRION
                                                                                                                                                produced                   by          the          pentose              pathway                is in            the
                                                                                                                                                cytoplasm.                  Thus              a shuttle               system             such          as that
                                  PROGESTEEONE                                -H            PREGNENOLONE                                        described               by Simpson                         and        Estabrook                (1969)                 is
                                                                                                                                                required              to transport                         the       reducing               equivalents
                                                                                                                                                into       this      organelle.
      FIG.      1. Possible        sites     of action         of cyclic      AMP on                                                                There    is some      evidence        in support    of                                                             a
steroidogenesis            in a hypothetical                 gonadal      cell. Solid                                                           mechanism      such    as that     suggested      by Uaynes
arrows       indicate        biochemical           reactions,        and the large                                                              and coworkers       for the action         of LH in ovarian
dashed       arrows      indicate        possible       effects     of cyclic      AMP
                                                                                                                                                tissue.           Phosphorylase                        activity             has      been        detected
on the steroidogenesis                     process.         Five of the dashed
arrows      indicate       that the possible             effects     of cyclic     AMP                                                          in      bovine           corpora                lutea            (Williams               et al.,         1961;
might      be mediated             through        protein        kinase,     but it is                                                          Marsh    and               Savard,     1964a;   Stansfield                                 and Robin-
also possible         that cyclic         AMP       influences        these steps in                                                            son,  1965;                  Yunis     and Assaf,       1970),                               luteinized
steroidogenesis            by a mechanism                not involving         protein                                                          rat ovaries                (Stansfield      and Robinson,                                   1965)       and
 kinease.             The symbol     ----p                                         indicates     transport                        of        a
                                                                                                                                                prepubertal                      rat         ovaries             (Selstam                 and          Ahren,
 substance             through   membranes                                          of the cell.
                                                                                                                                                1971;             Ahren     and                 Selstam,               1971;   Ahren     et al.,
                                                                                                                                                1973).             Furthermore,                        LU             has been     shown      to
is evidence                       supporting                        each                    of        these       proposals,
                                                                                                                                                increase            this        activity              in vitro            in incubating                   slices
and       at         the          present                   time                   it        is not             possible               to
                                                                                                                                                of bovine     corpora                                lutea       (Marsh       and                      Savard,
choose    any                     one of them                                 as the                   correct   one.                  In
                                                                                                                                                1964a)    and     whole                                 isolated        prepubertal                              rat
this regard,                       however,   it                              should                     be recognized
                                                                                                                                                ovaries            (Selstam                  and          Ahren,            1971;           Ahren               and
that      these  proposals                                  are       not mutually                                exclusive,
                                                                                                                                                Selstam,      1971;    Ahren   et al.,                                     1973).            It has also
and       it is possible                           that              cyclic AMP                                 may act by
                                                                                                                                                been    found     that the injection                                       of LU            into rats in
one       or more                  of these                  mechanisms.                                    The        evidence
                                                                                                                                                vivo will             cause an increase                             in the          phosphorylase
in favor                   of (or                 against)     these                                       hypotheses                  is
                                                                                                                                                activity             in the   prepubertal                               or          the luteinized
discussed                   below                 with     emphasis                                         on     the          data
                                                                                                                                                type    of ovary    (Stansfield                                     and Robinson,                        1965;
obtained                from             experiments                                    with            gonadal            tissue.
                                                                                                                                                Seistam    and Ahren,          1971;                                 Ahren   et al.,                    1973).
The        results     of some         important        experiments
                                                                                                                                                     In the experiments           on                                bovine  corpora                      lutea,
with        adrenal      cortex    tissue      are also      included,
                                                                                                                                                Williams             et al.            (1961)              reported               that      although                   a
since        the    control     of steroidogenesis               in this
                                                                                                                                                crude         pituitary                      preparation                    and       hCG              signifi-
tissue       has the same        general       features     as that    in
                                                                                                                                                cantly        increased                      phosphorylase                     activity,               a prep-
the      ovary             and       the          testis.
                                                                                                                                                aration            of luteinizing                         hormone                 was      ineffective.
                                                                                                                                                This         study              appears,                   however,                 to      have          been
Action              Via Increased                            Cofactors
                                                                                                                                                carried             out     with     only    a                        limited             number                  of
       Haynes                and         Berthet                    (1957)                       and          Uaynes           et al.           corpora              lutea,      and    when                           a large            number                  of
(1960)            were               the     first                   to propose                                 that        cyclic              corpora            lutea           obtained                 from          cows           in the         first         6
AMP            might                accelerate                         steroidogenesis                                     in the               months              of          pregnancy                    were          examined                    (Marsh
adrenal              cortex               by           increasing                            the           concentration                        and       Savard,            1964a),                 LU      was         found           to produce                    a
of a necessary                                cofactor                   in the steroidogenic                                                   small        (35           percent)                  but       significant                 increase               in
pathway.   Briefly,                             their                proposal   stated   that:                                         1)       phosphorylase                          activity.    The   luteal  phosphoryl-
ACTU     increased       the synthesis       of cyclic     AMP in                                                                               ase activity                 was           measured     in an assay     medium
the adrenal      cortex,    which      in turn   activated    the                                                                               containing                  5’-AMP,                  which    indicated                         that    this
phosphorylase                            system;                    2) phosphorylase                                       activa-              enzyme             was       similar               to the liver enzyme                            which      is
                                                                                         CYCLIC                 AMP          AND          STEROIDOGENESIS                                                                                                              41


only            partially                  activated                    by         the        nucleotide,                     and           corpora                 lutea          (Stansfield                  and            Robinson,                      1965),
differed        from      skeletal        muscle     phosphorylase                                                                          luteinized                 rat ovaries     (Deane,     1952;                                          Armstrong,
which       is completely          activated       by 5’-AMP.      This                                                                      1963;                Stansfield       and     Robinson,                                              1965)      and
was      confirmed         when        the    luteal   enzyme       was                                                                     rabbit               ovaries                  (Dorrington                       and                    Kilpatrick,
purified                and           characterized                             by Yunis     and                           Assaf            1967)              contain               little     or no                measurable                      glycogen.
(1970).                 The            stimulation                            of phosphorylase                                      in      Stansfield                 and Robinson                             (1965)                   found          that the
the         incubating                           slices                of          corpora                     lutea          was           injection                of LU into     rats                        in vivo                  caused          about   a
specific               for         LU-containing                                 hormones                      and        corre-            4-fold                increase                 in       phosphorylase                                 activity                 in
lated           with            an      increase                   in progesterone                               synthesis                  their              luteinized                       ovaries              4         h         later,             but        no
in these      slices.                       The addition of cyclic   AMP at a                                                               significant                     changes                in      glycogen                      concentration.
concentration                              of 2 mM to the incubating    slices,                                                             Ahren                  and           coworkers                      (Selstam                       and           Ahren,
however,                     did       not          cause              a rise              in phosphorylase                                 1971;              Ahren              and           Selstam,                 1971;                 Ahren              et al.,
activity,                  although                   it      did           cause             a small                increase               1973)              reported                 that        the       in vivo                injection                of LH
in progesterone          synthesis.       One                                               difference                    in the            or    the             in vitro  incubation    of                                         prepubertal      rat
LU and        nucleotide        incubations                                                     was the                   inclu-            ovaries               with   LU or FSU caused                                           a rise of ovarian
sion    of                 0.02             M caffeine                           in         the         cyclic              AMP             phosphorylase                            activity.               The              phosphorylase                            of
medium.                     This            methylxanthine,                                       in     addition              to           this         prepubertal                     ovary            was        reported      to resemble
its inhibitory         effect   on cyclic       nudeotide          phos-                                                                    that          of skeletal                   muscle            and         the increase      was due
phodiesterase,          has also been        reported       to stimu-                                                                       entirely               to a shift  from                          the 5’-AMP                           dependent
late     phosphorylase           phosphatase           (Sutherland,                                                                         b form,                 to the 5’-AMP                            independent                           a form              of
1951),       which      may    account        for the       small     de-                                                                   the          enzyme.                  The            addition                of        dibutyryl                  cyclic
crease               in         phosphorylase                             activity        actually                             ob-          AMP              at     concentrations                           from              1-25               mM          to      the
served                in         the    cyclic                         AMP       incubations.                                 The           incubating                          ovaries             was          more                    effective                  than
presence                   of caffeine                      does            not,         however,                    seem           to      either             LU         or      FSH.            Epinephrine                        at a concentra-
account                     for            the             lack             of      response                         of      this           tion          of        10          mM         also           increased                      the       percent                 of
phosphorylase                                system                    to        cyclic     AMP,                          for in            phosphorylase                          a.
other   experiments                                    (Marsh,      unpublished                                             data)                   Thus            it         appears             that         LU            and             perhaps               FSH
the omission        of                           caffeine      from     the cyclic                                          AMP             can          stimulate                 phosphorylase                              in         several            ovarian
treated               samples                    eliminated                         the           slight             drop           in      preparations,    but it seems     that                                                   this effect     may
activity,                  but         the          exogenous                            cyclic            nucleotide                       not be essential    for an increase                                                     in steroidogene-
still      did         not         increase                  the        phosphorylase                             activity.                 sis.         A        similar            situation                  exists                   in     the         adrenal
A larger                   concentration                           of cyclic                    AMP              (0.02     M)               cortex.                   Uaynes                     (1958)        demonstrated                                         that
has also                    been      tested                      on these                     bovine              corpora                  ACTU                   stimulated                       phosphorylase           activity                                    in
lutea           in the               absence                 of        caffeine                and         again            there           bovine     adrenal      cortex,      but Ferguson         (1963)     and
was     a             stimulation                            of         progesterone                             synthesis                  Kobayashi          et al. (1963)         found       no stimulation
without                  an effect                          on          phosphorylase                              activity                 by ACTU             in rat      adrenal      cortex      tissue     even
(Savard               et al.,              1965).                                                                                           though      this hormone            produced        its usual    stimu-
         This           discrepancy                           has            never              been             resolved,                  lation    of corticosteroidogenesis.
but it has                         been           suggested                      that   adding   LU to                                              It       has           been            found              that                 the          addition               of
incubating                        corpora             lutea                 slices    may    be a much                                      NADPU                to             incubating              slices   of bovine     corpora
better               way              of         increasing                        intracellular                          cyclic            lutea            (Mason                et al.,           1962;     Savard   et al., 1963;
AMP              than             adding              the           nucleotide                         exogenously.                         Savard                 and    Casey,      1964;                              Mason      and     Savard,
Nevertheless,                              this  inability                            to          activate    phos-                         1964b;                 Armstrong,         1966),                              to slices     of rabbit
phorylase                       by           a concentration                                       of exogenous                             corpora                 lutea    or interstitial                               tissue   (Dorrington
cyclic               AMP       which                              stimulated                        progesterone                            and    Kilpatrick,      1966b)        or to homogenates         of
synthesis              indicated                             to       us that                     phosphorylase                             whole     rabbit   ovaries       (Scoon    and Major,    1972),
activation                      might             not             be        an        essential                  effect             of      caused     a marked        increase     in progestin    biosyn-
LU         in      stimulating                      progesterone                                synthesis.                   This           thesis.               In addition,    Savard      et al. (1963)                                               demon-
was             supported                        by    the     fact                           that      about                   10          strated                that  the bovine      corpus     luteum                                              had high
percent                    of        the         corpora                     lutea             tested                showed                 concentrations                           of the               NADP                dehydrogenases                               of
little          or      no           increase                in terms                    of       phosphorylase                             the           pentose                  pathway.                     It         became                     apparent,
activity              while            they           exhibited                       a good             response                   to      however,                     that        the         effect         of        NADPU                       was         quali-
LU      in            terms     of                   an           increase                    in progesterone                               tatively       quite                    different    from     that                                  of LU. The
synthesis                (Marsh                     and             Savard,                    1964a).     This                      is     first    indications                         of this difference                                      came  from
also            supported                        by           the            finding                    that           bovine               the          study            of      Savard            et al.           (1963)                    where          it was
42                                                                                                                              MARSH



found              that           the          addition                    of        LU        to luteal                 slices,           preparations                         into       rabbits                and          rats      will          cause             a
incubating                      in      the        presence                      of       optimal                 amounts                  decrease               in           ovarian             cholesterol                        (Claesson                 and
of NADPU,       caused     a further                                                   increase  in proges-                                Uillarp,   1947;     Levin     and Jailer,                                                  1948;      Bell et
terone synthesis.      This should                                                      not have occurred                                  al., 1964;   Herbst,     1967;    Armstrong,                                                     1968)     and
if the              effect      of LU                           was              mediated                       solely    by               that       nearly              all of this              decrease                can          be accounted
NADPU.                   Dorrington                             and              Kilpatrick                       (1966b)                  for      by      a decrease                     in cholesterol                         ester         (Claesson
also        observed                         additive                      effects                of          maximally                    et al.,          1948,      1953;                       Uerbst,                 1967;   Armstrong,
effective                  concentrations                                   of         LU          and            NADPU.                   1968).           Furthermore,                           Moyle                 et al. (1973a)       have
The effects    of                            LU and                    NADPU     on incubating                                             shown      that                  the addition       of LU or cyclic   AMP
slices of bovine                              corpora                  lutea also appeared                                           to    to incubating                       Leydig    tumor    cells causes a rapid
be       different                    in         terms            of        precursor                      utilization.                    (10        mm)             conversion                     of          ester         cholesterol                     into
LU increased        the incorporation       of                                                           [i1 4C] ace-                      free       cholesterol.                       This        effect               could          come             about
tate into    progesterone        to a greater                                                            extent  than                      either           by             a       stimulation                       of         the          cholesterol
the            incorporation                                          of              [73         U]      cholesterol.                     esterase             or         by       an        inhibition                  of      the        cholesterol
NADPU,                     on          the         other               hand,                did         not        increase                ester         synthetase,                     and evidence                          for      both           sites         of
the incorporation                              of [114       C] acetate,                                    but mark-                      action          has been                    presented.
edly enhanced                              the incorporation          of                                  [7-3U]cho-                             Behrman                        and             Armstrong           (1969)                            demon-
lesterol              into              this            steroid                  (Savard                  and           Casey,             strated      that                   the       in      vivo     administration                                of LH
1964;              Mason                   and          Savard,                      1964b).                  Finally,                it   increased    cholesterol                                esterase  activity                           in lutein-
became               apparent                      from              the          work             of      Armstrong                       ized ovaries      of rats.                              No direct     effect                          of cyclic
(1966)               on     the                    bovine                       corpus                  luteum       and                   AMP            (0.05             mM)            or      theophylline                         (1      mM)             was
Halkerston                       (1968)                  on          the         adrenal                  cortex              that         observed,               however,       when                        these             substances       were
exogenous                        NADPH                         probably                       acted               only           on        added     to           the cholesterol                           esterase              assay    mixture.
damaged                   cells             while              LU           and             ACTU                 acted           on        Flint         et al. (1973)                     have           also       reported                 that        cyclic
intact         cells.                                                                                                                      AMP           had no direct            effect                            when       added       to the
       In      regard                 to         the          endogenous                          concentration                            assay         system    for cholesterol                                    esterase      activity       in
of NADP    and NADPU         and the effect    of LH, it                                                                                   rabbit          ovarian    interstitial                               tissue.      Another        piece
has been reported   that this gonadotropin         has no                                                                                  of evidence        indicating        that      LH activates       the
effect  on    the concentration        of   either     the                                                                                 cholesterol    esterase       enzyme        is that   of Behrman
reduced               or         the          oxidized                     form              of        this       pyridine                 et al. (1972),      who showed            that    the administra-
nucleotide                        in bovine                        corpora       lutea        (Marsh,                                      tion       of an LU                    antiserum                to pregnant                       rats      in vivo
1968)               or           luteinized                       rat    ovaries       (Flint      and                                     resulted              in a              marked   decrease                                  in cholesterol
Denton,                  1970).                Furthermore,                             the            latter       investi-               esterase             activity            and a reduction                                  in progesteron
gators             also found                          that       glucose raised   the intra-                                              secretion.
cellular              concentration                               of NADPU       in slices    of                                                It is possible                           that       this         stimulatory                        effect          of
luteinized                      rat         ovaries,                but               had   no effect                            on        LU on cholesterol        esterase    is secondary        to the
steroidogenesis                              in this              tissue.               It was concluded                                   action   of this hormone        on steroid    synthesis.      An
(Flint         and          Denton,                  1970),                  therefore,                     that it was                    enhanced     conversion       of cholesterol       into    preg-
unlikely                 that           LU         brought                    about     its                increase   in                   nenolone                  might                remove                  a hypothetical                             feed-
steroidogenesis     solely      by increasing                                                           the produc-                        back     inhibition                           of the    esterase                           enzyme.                  The
tion      of NADPU.        Hall    (1971)                                                         criticized      this                     evidence       on this                        point  is equivocal.                            Behrman                  et
interpretation                             saying              that             it     did         not           take         into         al.      (1970)                  concluded                       that            the          LU-induced
account               the         possibility                    that an increased    rate of                                              depletion                 of      cholesterol                ester    was not due                                   to     a
utilization                     of NADPH                         might  balance    an LU-in-                                               secondary                      effect       of             LU      on   steroidogenesis,
duced          increase                    in the             rate         of its production,                                  and         since         they             could           observe                 the      action              of       LU          on
thereby                   maintain                      an           unaltered                         steady                state         cholesterol                    ester         depletion                 when           steroidogenesis
concentration                                of          NADPU.                         This     alternative                               was        blocked                      by         aminoglutethimide.                                     On         the
seems       theoretically                                possible,                     but until       there                          is   other          hand,                Flint          et    al.      (1973),                  working                with
more           information                              on       how                 NADPH                      is utilized                the same      tissue,  found    that   the administration
by the side-chain      cleavage                                                  system      in situ,                         it is        of aminoglutethimide          or cycloheximide          in vivo
impossible    to reach     a firm                                                 conclusion        on                         this        blocked     the effect     of LU on the depletion             of
matter.                                                                                                                                    cholesterol                    ester.
                                                                                                                                                Goldstein                     and          Marsh             (1973)                   have           assessed
Action              Via Increased                             Substrate                                                                    the effect               of LU and cyclic      AMP on incubating
       It     is     well              known                   that             the         injection                   of      LH         slices  of             bovine  corpora    lutea   and found      that
                                                                                  CYCLIC                 AMP            AND       STEROIDOGENESIS                                                                                                                   43


LU       and        cyclic               AMP          treated               tissues                had        slightly               suited              in         an         increase                in        cholesterol                          esterase
higher    values     of cholesterol       esterase                                                        activity.                  activity                 with     a               concomitant                          transfer                 of 32P
Moreover,      homogenates        of this tissue                                                      responded                      labelled                  phosphate                     to the                       protein                  fraction.
to cyclic       AMP      and    ATP         with     a significant                                                                  These             data               led         Trzeciak                and               Boyd            (1974)                to
increase    in cholesterol        esterase       activity     (Gold-                                                                 propose                  that             the       activation                       of         adrenal               cortex
stein,   1973).     This     effect        of cyclic      AMP        in                                                              cholesterol                      esterase                by      ACTU                     involves               a cyclic
homogenates                             and        the        report               of         Moyle                et       al.      AMP-dependent                                    protein           kinase                  and       the         transfer
(1973a)                 of         a stimulation                       of      cholesterol                      ester                of a terminal                             phosphate                group     from   ATP                                  onto
conversion                        into   cholesterol                         by cyclic                       AMP      in             the cholesterol                              esterase              molecule.      There                                  have
isolated            Leydig                cells       suggested                   that          the       effect            of       been   two     other    reports                                    of a direct                       stimulation
LU          and          cyclic                 AMP          on        cholesterol                           esterase                of cholesterol       esterase                                    activity   by                      cyclic     AMP,
might              be         mediated                   through                  a protein                        kinase            ATP and protein     kinase    in idrenal                                                            (Naghshineh
phosphorylation                                 reaction,              such              as        that            which             et al., 1974)   and   adipose     tissue                                                          (Pittman      and
acts         on         the            adipose              tissue           triglyceride                          lipase            Khoo,            1974).
(Uuttunen                         et     al.,      1970;             Corbin                et         al.,         1970;                The            other                 possible               way           in           which            LH          could
Steinberg              and     Huttunen,        1972).        Cyclic    AMP-                                                        control                cholesterol                        ester          concentrations                                  is via
dependent                protein        kinase     activity         has  been                                                       the           inhibition                    of      the cholesterol   ester                                        synthe-
detected             in bovine      corpora     lutea      (Goldstein      and                                                      tase          system              and            this has been explored                                           by Flint
Marsh,             1972,     1973;       Menon,      1973),       rat ovaries                                                       et      al.      (1973).                   These            investigators                           showed                 that
and         Graafian                   follicles            (Lamprecht                        et al.,              1973;            the     administration           of                               LU to rabbits                                   in       vivo
Tsafriri            et        al.,         1972b;             DeAngelo                        et      al.,         1973;            decreased        the cholesterol                                   ether synthetase                                      activ-
DeAngelo                  et al.,               1974)         and rat testes  (Reddi                                       et       ity       in      ovarian                   interstitial                 tissue              and           this        effect
al., 1971;                Means                 et al.,       1974).  This enzyme                                        has        was      mimicked       by cyclic                                            AMP      in vitro.    The
also       been          partially                purified             from             bovine               corpora                inhibition        of this  enzyme                                              system     by LU or
lutea             (Menon,                    1973;       Goldstein                          and                Marsh,               cyclic     AMP seems to be the                                               result   of an increase
 1973)            and rat              testes     (Reddi      et al.,                     1971).                                    in      steroidogenesis.                                 Progesterone                             and          20a          hy-
     Cholesterol                      esterase         was                   partially                       purified               droxypregn-4-en-3-one                                             are            synthesized                        at          in-
from      the                 soluble         fraction                      of bovine                          corpus               creased      rates    in                           interstitial                   tissue     after                  LU           or
luteum             homogenate                         by ammonium                               sulfate              frac-          cyclic     AMP      treatment       and these      steroids     were
tionation             (Goldstein,                       1973).    The                           addition                 of         found      to exert      a direct    inhibitory     effect    on the
cyclic            AMP    dependent                            skeletal                muscle    protein                             synthetase      system.        It was also found         that if the
kinase            to this partially                          purified               luteal   cholester-                             effect      of cyclic       AMP      on steroidogenesis          was
ol esterase                    increased                  the esterase                        activity,    but                      inhibited                  by cycloheximide                                        or aminoglutethi-
cyclic    AMP                    or ATP                  did not cause                          any further                         mide      then              the inhibition                              of       the synthetase                            was
increase      in activity                          (Goldstein,                      1973).                The           role        also      lost.             This               result            is in             keeping       with    the
of     cyclic    AMP                             and      protein                     kinase                in           the        interpretation                             that     LH          inhibits             cholesterol      ester
activation                   of        luteal         cholesterol                    esterase                  is thus              synthetase                       by              increasing                  the             production                         of
still      uncertain                    at this          time.         A thorough                         study             of      progestins.                     It seems,                 then,          that              LU       increases               the
the        role         of         cyclic           AMP           in        the          stimulation                        of      level           of         free             cholesterol                      at            the       expense                    of
cholesterol        esterase      of adrenal      cortex  tissue     has                                                             cholesterol                       ester             in       rat     and                    rabbit    gonadal
been      carried      out    (Simpson      et al., 1972;        Boyd                                                               tissues     by                   both              an       activation                        of cholesterol
and Trzeciak,            1973;     Trzeciak    and Boyd,        1973,                                                               esterase                  and          an          inhibition                    of         the         cholesterol
1974).       In brief,     it was found      that stress    induced                                                                 ester          synthetase.
by      ether           anesthesia,                   which            is known                       to elevate                         The             importance        of this                               action   of                   LU in the
plasma             ACTII       concentrations,            increased       protein                                                   control               of steroidogenesis                                     in gonadal                      tissue is
kinase            and     cholesterol          esterase      activities      in rat                                                 difficult              to assess.                    It is possible                      that by                  keeping
adrenal            cortex.       The addition           of cyclic       AMP and                                                     a greater                proportion                       of the                      cholesterol                    in its
ATP           in         vitro            also           increased                  the            activity                 of      free           form,                 LU           regulates                  a         limiting                   step          in
cholesterol                       esterase            preparations                        from               rat        and         steroidogenesis.                              On         the      other            hand,             this         may           1e
bovine             adrenal                 cortex    and                    these    effects     were                               just   a supporting                               effect,                supplying                       more             sub-
dependent               upon                the presence                      of cyclic      AMP-de-                                strate   when     the                        steroidogenic                    pathway                      has           been
pendent                  protein                 kinase.             Preincubation                               of a               accelerated                      by         an action              of the                  gonadotropin                         at
partially               purified                bovine            adrenal       cortex                        choles-               another                and   more                       crucial            step.  In                    the         bovine
terol        esterase                   with          [‘y-32P]               ATP,               cyclic              AMP             corpus               luteum,      at                   least,            the effect                       of        LU on
and        cyclic              AMP-dependent                                protein                   kinase             re-        cholesterol                      ester            storage          would                   appear              to        be of
44                                                                                                                   MARSH



relatively                 minor     importance,                               since    it has                been           been     found    in the mitochondrial                                                           and       cytosol
reported                  that   this tissue,                      which            is capable                 of a          fraction     of bovine  adrenal    cortex                                                    tissue        (Kan et
marked     steroidogenic         response     to                                           LU, has large                     al., 1972;            Kan          and   Ungar,                          1973;                 Ungar     et al.,
amounts       of free     cholesterol       and                                            small   to un-                    1973).     It         has          a specific                          binding                  affinity    for
measurable        amounts      of cholesterol                                                ester   (Zim-                   cholesterol                 and          stimulates                           the            activity             of        a
belman             et al., 1961;                   Uafs            and         Armstrong,                    1968;           cholesterol              side-chain                        cleavage                    preparation                     of
Seifart           and Hansel,                    1968).                                                                      adrenal         mitochondria.                              Ungar              et       al.      (1973)            have
                                                                                                                             proposed             that         this         protein                  represents                      a choles-
                                                                                                                             terol    carrier   protein                           which                  can       transport                 cho-
Action                Via         Increased                   Transport                    of
                                                                                                                             lesterol      from     the                         cytoplasm                        into      the              mito-
Cholesterol
                                                                                                                             chondrion,                  but          no           direct                  evidence                   for        this
        Since             the          cholesterol                       side-chain                    cleavage              function       is available                         at this time.    This   choles-
system             has          been         shown             to         be     localized                  in the           terol    binding     protein                          resembles   the regulatory
mitochondria                           of     bovine     adrenal      cortex     (Ual-                                       protein       proposed        by Garren       et al. (1971),       but
kerston       et                al.,        1959,     1961),     bovine      corpora                                         this     hypothesis        required      that    ACTU      increase
lutea     (Hall                  and          Koritz,     1964;      Yago      et al.,                                       the     synthesis       of this      protein.       Ungar      et al.
1967),            rat       testis          (Toren             et al.,           1964;               Drosdow-                (1973)        have     reported,      however,        that    ACTH
sky    et          al.,         1965),     mouse                      Leydig     cell tumors                                 does    not     change                    the          level                of      this            cholesterol
(Moyle              et          al.,   1973b)                      and     human      placenta                               binding     protein.
(Mason       and                 Boyd,           1971),             and since    cholesterol                                        Mahafee              et al. (1974)                                have                demonstrated
is synthesized                     and          stored             outside   this organelle,                                 that      ACTU              and   dibutyryl                              AMP                  treatment                of
there           must            be      a migration                       of     cholesterol                   into          hypophysectomized                                  rats increased       the                               level       of
the       mitochondria                          before              it can be converted                                      free    cholesterol                      in        adrenal    mitochondria                                          and
into       pregnenolone.                          There             has been a report                                of      the conversion                     of this                   steroid   into   pregneno-
cholesterol                     sidechain               cleavage                activity               in     both           lone. They     also                showed                    that this stimulation                                     of
the microsomes                         and the mitochondria                                   of bovine                      cholesterol             accumulation                               in mitochondria                                  was
corpora    lutea                     and rat luteinized                               ovaries     (Flint                     probably             not secondary                               to the stimulation                                   of
and           Armstrong,                      1971),               but         this         will        be        dis-       steroidogenesis                 at                 some                other               site   because
cussed           later          in this        section.                                                                      ACTU        and         dibutyryl                    cyclic             AMP              promoted       this
        The       transport                  of cholesterol                       into          mitochon-                    accumulation                      even              when                    aminoglutathimide
dna       might     be               a limiting    step in steroidogenesis                                                   was      administered                   in doses                                 known       to                block
and       therefore                   could     be another    candidate                                           for        steroidogenesis.                     Cycloheximide                                    treatment                        of
the   site   of tropic                           hormone                    action.      Although                            the rats also               did not prevent                                   the mitochondrial
there    has been    very                         limited                experimentation          on                         accumulation                  or cholesterol                                  brought    about                         by
cholesterol                     transport               in      gonadal                tissues,              there           ACTU,           indicating                     that             this             protein                synthesis
has       been            a considerable                       amount                 of        work         done            inhibitor            blocks              the           stimulation                             of       steroido-
on this possibility        in the study       of the mecha-                                                                  genesis        at     some          later             step             in        the          process.             The
nism of action      of ACTH         and cyclic    AMP in the                                                                 authors’            interpretation                              of       the            data            was       that
adrenal cortex.       This    possibility      was first pro-                                                                ACTU   and cyclic                         AMP               increased       the accumula-
posed           by      Uechter    (1955)                           and then     by Garren                                   tion of cholesterol                                   in      the     mitochondria        by
(1968).              In the latter   work,                          it was suggested    that                                 increasing             the       ratio                     of          free     cholesterol      to
such          a translocation                           of      cholesterol                      might             in-       esterified           cholesterol                      in        the        cell. It is possible,
volve           the         synthesis                  of      a         regulatory                    protein,              however,            that     ACTH,     via cyclic  AMP, activated
since          cycloheximide,                           an          inhibitor                   of     protein               a carrier             protein,     such    as that   described    by
synthesis,     blocked                         the          acceleration        of steroido-                                 Ungar         et al. (1973).
genesis     by ACTH,                           and           increased       the accumula-                                          A modification                         of      this           latter         type            of propos-
tion     of cholesterol                          in         extramitochondrial             lipid                             al was         suggested                 by        Simpson                       et al.         (1972)              and
droplets.                It was         proposed                   that        AC1’H,                via cyclic              Jefcoate      et al.              (1974).      They     found   that    ether
AMP,             enhanced                    the      synthesis         of                      a      protein               stress   increased                 the initial     rate of pregnenolone
which           facilitated                 cholesterol         transport,                            and that               formation     from                 endogenous                            cholesterol                      in adre-
when           cycloheximide                          inhibited                protein               synthesis               nal mitochondria                      isolated                         from      these                   animals,
it blocked       cholesterol                                  transport                 and             the     in-          but there      was no effect     on the initial    content        of
crease   in steroidogenesis                                    (Garren,                1968;             Garren              cholesterol      in these   organelles.   The ether        stress
et al.,        1971).                                                                                                        also induced        changes    in the type      II difference
        A heat            stable            cholesterol                   binding           protein               has        spectra     of cytochrome        P450   which      were inter-
                                                                               CYCLIC                AMP           AND         STEROIDOGENESIS                                                                                                  45


preted           as indicating                       an increased                       amount               of the              labelled   cholesterol                           is uniformly        diluted        by                        the
cholesterol-cytochrome                                     P-450               complex.                  It was,                 endogenous        pools,                        then    the specific        activity                            of
therefore,        proposed                           that    only              a fraction                 of the                 this substrate       would                          be about      four                    times           lower
total      mitochondial                                cholesterol                 was                available                  in the     mitochondrial                              incubations                        than            in the
for        side-chain                      cleavage,                and            that           stress             via         microsomal                     ones.            Thus           the      same                 amount                of
ACTU             increases                    that          fraction              which              is active.                  radioactivity                      in     the       products                of         the      sidechain
This          increase                could           be      due       to        the         transport               or         cleavage              reaction              would          represent                    four         times         as
binding              of cholesterol                        to the          side-chain                  cleavage                  much           cleavage                 activity          in     the        mitochondria                           as
cytochrome                          P-450            from           other           intramitochon-                               in the         microsomes.
dial          sites.    It is also                   possible          that these   stress-in-                                        Moyle   et al.                      (1973b)               determined                      the       locali-
duced            changes      may                    result         in an increased       entry                                  zation     of the                        cholesterol                 sidechain                       cleavage
of      cholesterol                    into           the        mitochondria                           in        vivo.          enzyme               system              and       the     endogenous                         cholesterol
This          effect             of        stress          was         found             to      be       readily                in     mitochondria                        from          Leydig             tumor                cells       and
inhibited         by                  cycloheximide                          indicating      that   a                            found          that          the        enzyme           activity           was          localized             on
labile    protein                     may      be involved                       with   this trans-                              the      inner         membrane                     of the        mitochondria,                           while
port       or binding.                                                                                                           essentially              all of the cholesterol                                   was associated
        The          effect           of      gonadotropins                             or     cyclic         AMP                with      the         outer    membrane.      This                               indicates    that                  a
on       cholesterol                   transport                 into        mitochondria                            has         limiting              step       in steroidogenesis                                could    be the
not      been     studied                       very    extensively                             in     gonadal                   availability                 of substrate     cholesterol                            in this tissue
tissues.      Flint     and                     Armstrong         (1971),                             using    an                as well.    Robinson      and    Stevenson                                            (1973)     have
assay           involving                 the          production        of l4                               C] iso-             also reported       in an abstract      that                                       the treatment
caproic              acid              from             [2614     C] cholesterol,                                 re-            of     superovulated                            immature          rats                     with      hCG
ported               that         there              was      about                twice              as much                    causes      a marked                        increased       in the                      transport       of
side-chain                    cleavage               activity       in             bovine               corpora                  cholesterol          into                     the     mitochondria                            obtained
lutea         microsomes                       as in mitochondria.                                    Rat         lute-          from   the luteinized                           ovaries.
inized           ovaries               were            also         found               to     have          about                  Thus,    although                            an effect              of          LH          and cyclic
one   quarter   as much       of                                 this        activity       in micro-                            AMP      on cholesterol                             transport                    into          mitochon-
somes     as in mitochondria.                                              This      suggests     that                           dna       remains                   to be examined           thoroughly          in
there      may                be no           requirement                     for a transport         of                         gonadal       tissue,                the studies    on the adrenal        cortex
cholesterol                     into          mitochondria                       in luteal    tissue.                            implicate        this              process    in the control      of steroido-
We       have          been           unable,               however,                to        confirm               this         genesis.
distribution           in                  bovine     corpora                       lutea,    using     an
assay        involving                        the    conversion                          of    [414    C]
                                                                                                                                 Action           Via Increase                      Side-Chain
cholesterol          into                   labelled     steroid                     products       (Car-
on,       1973).               In      our          hands,           the          cholesterol                     side-          Cleavage              Activity

chain  cleavage    activity      shows                                       the same                 distribu-                       Cyclic            AMP              could     increase              the conversion        of
tion in the different       fractions                                         of luteal               homoge-                    cholesterol             into             pregnenolone                   by a direct    action
nates    as the marker                                 enzyme     for mitochondria,                                              on       the         side-chain                 cleavage              enzyme                  system           by
succinic    dehydrogenase.                                  There    was no correla-                                             activating,    or increasing                              the synthesis                          of one of
tion    between                        the           cleavage                 activity                and        the             the components         of this                           complex.     Such                        an effect
microsomal                          marker              enzyme,                   NADPU                       cyto-              of      cyclic              AMP           was          proposed                   to         explain           its
chrome     C reductase.        It is unknown        at this                                                        time          stimulation                   of     the conversion                      of labelled      cho-
if this discrepancy         between     the results      of                                                       Flint          lesterol             into          pregnenolone                       in various     adrenal
and Armstrong          (1971),      on the one hand,                                                                 and         cortical       mitochondrial         preparations       (Roberts        et
the      data               of        Caron     (1973)                         and             most   other                      al., 1965,         1967;     Roberts       and Creange,          1968).
investigators,                        on    the   other                       hand,             is due      to                   Koritz       et al. (1968),         however,      were    unable        to
methodological                          differences,                        but it seems     that                                substantiate          this proposal       and suggested        instead
the former                       workers        did              not         take into account                                   that       the         increased                 pregnenolone                          accumulation
the     endogenous                            levels          of cholesterol                           in these                  was due to an inhibition                 of the conversion                                                      of
fractions       when                         they           calculated       the                      levels  of                 pregnenolone          to progesterone             rather    than                                               an
side-chain                    activity.               We         have             found               that           the         increase           in   pregnenolone             synthesis.                                                  The
mitochondria                           isolated                from               homogenates                             of     t53j3hydoxysteroid                  dehydrogenase           and                                               the
bovine               corpora                lutea           contain               more            than             four          A53ketosteroid                             isomerase                 enzymes                   which          are
times           as      much                endogenous                       cholesterol                     as      the         involved              in this            reaction          appear                to be present                     in
microsomes                          (Caron,             1973).               If     the          exogenous                       both           the          mitochondria                        and         the              microsomes
 46                                                                                                                    MARSH



(Sulimovici                      and      Boyd,                    1969;   McCune                      et          al.,        tion       of enzyme                  activity              of the           order           of 20 to 70
1970).      This                inhibitory                     effect    of cyclic                   AMP            on         percent.             The          stimulation                         was         completely                    de-
the        pregnenolone                         to      progesterone                    reactions                  has         pendent      upon      the                     presence                  of ATP                 indicating
been          confirmed                        by       several           investigators                        using           that    a phosphorylation                                       was      probably                 involved.
adrenal       and   ovarian     preparations                                                  (Sulimovici                      This        in       vitro            effect               of      cyclic              AMP             on       the
and     Boyd,     1969;     McCune        et                                    al.,          1970;       Suli-                reconstituted               system    may   represent                                               a physio-
movici                and       Lunenfeld,                      1971;          Snnivasan         et al.,                       logic     action          of the cyclic   nucleotide,                                               but there
1973),                and       it seems                 that      the        cyclic     nucleotide                            will     have           to be further     confirmation                                                 of this
inhibits               the     5-3(3-hydroxysteroid                                           dehydroge-                       type of activation                          in situ             in whole               mitochondria
nase,            at      greater       than      physiologic                                   concentra-                      and whole     cells.
tions,           by         competing                  with        NAD          for          the     enzyme.
    Similar                  findings       have                 been     reported    in gona-
dal    tissue                   experiments.                        lchii      et al.   (1963)                                 Action     Via          Increased                   Efflux     of
                                                                                                                               Pregnenolone                From                  Mitochondria
suggested                    that         LU          itself       had        a direct               stimula-
tory        effect            on         the      side-chain              cleavage                 system              of              Konitz        and         Hall            (1964)               observed                that         preg-
the bovine                    corpus  luteum,      but this                              could    not be                       nenolone               inhibited                     its         own             synthesis                  from
confirmed                     by some     of these     same                               authors    in a                      cholesterol                in         an     extract                  from           adrenal                mito-
later        publication        (Yago    et al., 1967).                                         Sulimovici                     chondria,              and        since              it was   believed     that                                 the
and          Boyd        (1968)     then    reported                                          that     cyclic                  pregnenolone                     must              leave  the mitochondria                                        to
AMP both      stimulated                                  the conversion                        of choles-                     be converted         into      progesterone                                                 (Beyer      and
terol  to pregnenolone                                     and inhibited                      the conver-                      Samuels,      1956),      it was suggested                                                   that    ACTU
sion of pregnenolone                                    to progesterone.                         When    the                   might    control      steroidogenesis       by                                              determining
total    amount                   of pregnenolone     and progesterone                                                         the     egress    of                  pregnenolone                         from              the            mito-
synthesized                     in the cyclic     AMP incubations                                                was           chondria       (Koritz                    and    Hall,                   1964;              Koritz            and
compared     with    that    in the controls,     however,     it                                                              Kumur,       1970).     It was also reported        that   sub-
was apparent      that    there    was no overall     stimula-                                                                 stances     which     cause   mitochondria      of steroido-
tion        of        the      side-chain                  cleavage            activity               and         the          genic   tissues     to swell   stimulated    pregnenolone
increased       accumulation                                      ofpregnenolone                            was                synthesis,            while   ATP     which      inhibited                                           swelling
probably          due      to                             the     inhibition                         of      the               decreased            this stimulation       (Uirschfield                                            and Kor-
A533hydroxysteroid                                         dehydrogenase                            (Sulimo-                   itz, 1964,           1966).
vici and Boyd,                           1969;    Sulimovici                    and           Lunenfeld,                               There        are         several             pieces              of evidence,                       how-
1971;   Srinivasan                         et al., 1973).                                                                      ever,           which           indicate               that             this   hypothesis                           is
        Recently                    we         have        examined                    the         effect              of      probably             not         correct.                  First          of      all,        there           is a
cyclic        AMP,                    protein       kinase       and                          ATP         on a                 5-313-hy     droxysteroi        d dehydogenase                                                 present              in
reconstituted                          cholesterol         side-chain                                cleavage                  mitochondria             (Sulimovici       and                                       Boyd,                 1969;
system         from                   bovine       corpora        lutea                            and     have                McCune             et al.,        1970;             Sulimovici                    and Lunenfeld,
observed                a direct                 stimulation                   of       this          enzyme                   1971;           Srinivasan            et           aL, 1973)                     which   can con-
activity               (Caron                  et al.,       1975).                    The           cleavage                  vert      pregnenolone                      into           progesterone.                       Secondly,
system                was            solubilized                       from         corpora                    lutea           Simpson    et al. (1972)      reported                                         that    the           amount
mitochondria                        by            phospholipase                    A treatment                                 of pregnenolone          present       in                                      freshly               isolated
and     the                 cytochrome                   P450                  component       was                             adrenal            mitochondria                            from           stressed                  rats      was
isolated               from              the          other            components                         of      the          greater       than    that     observed                               in the         mitochondria
system                by column                       chromatography.                              The          cho-           from        cycloheximide-treated                                        rats,        even     though
lesterol               side-chain                    cleavage       activity                       was         then            there       was      increased                    cholesterol                  side-chain                  cleav-
reconstituted                     using    this                    isolated      cytochrome                                    age      activity          in the mitochondria                                     obtained                 from
P450        and               the non-heme                         iron     component       and                                the       stressed           animals.  Thirdly,                                  the    amount                     of
flavoprotein                        component                    of      adrenal               cortex             tis-         pregnenolone                     required         for                     this       inhibition                     is
sue        (adrenodoxin                         and adenodoxin                             reductase).                         greater      than                the      physiologic                             concentrations
The          proportions                        of these    three                        components                            which           occur     in           mitochondria         (Simpson     et al.,
were         arranged                    so that               the activity    was                    propor-                  1972).          Fourthly,               when      pregnenolone       was made
tional          to the                    amount                 of cytochrome                           P-450                 to accumulate                    in mitochondia,                                  by        blocking             its
present.               When              cyclic          AMP,            ATP          and          a protein                   conversion              to progesterone        with                                  cyanoketone,
kinase           preparation,                        partially            purified                 from         cor-           it did not             affect  the stimulation                                         of      side-chain
pora     lutea,      were     added     to this    reconstituted                                                               cleavage           activity            caused               by the             action           of endog-
cleavage      system      there     was a consistent        stimula-                                                           enous           ACTH             in        vivo        (Simpson                    et        al.,      1972).
                                                                                 CYCLIC                  AMP        AND        STEROIDOGENESIS                                                                                                                47



Similar           results               were              obtained              by Farese                   (1971)               incubated                        slices           of         bovine          corpora                   lutea,             Shin
using           incubated                      rat          adrenal              sections,                  ACTU,                and       Sato                 (1971),             using          incubated                 mouse                  Leydig
and      cyclic          AMP.                                                                                                    tumor                cells,              and           Tsafriri            et      a!.      (1973),                   using
                                                                                                                                 isolated                   Graafian                    follicles,               found             that             actino-
                THE          ROLE   OF PROTEIN                                  SYNTHESIS                                        mycin                D blocked                          the       action            of      LI-I and                 cyclic
                             IN THE STIMULATION                                     OF                                           AMP                 on         steroidogenesis.                            On        the          other               hand,
                                 STEROIDOGENESIS
                                                                                                                                 Gorski               and Padnos                          (1966)   reported                            that actino-
        Concomitant                             protein                   synthesis                 has            been          mycin                D had no                          effect   on the LH                             stimulation
shown to be required                                        for the actions                         of ACTH,                     of      steroidogenesis                                 in      incubated                 slices              of rabbit
LU and cyclic  AMP                                        on steroidogenesis                           in their                  ovary.               In regard                    to the          studies            on     corpora                   lutea,
respective               target               tissues.             I will       not       go into              detail            further                   experiments                        have    shown    that    this                                   in-
on this           matter,                  since             it was             covered                  by Wicks                hibition                   of the LH                     stimulation       by actinomycin
(1974)            in a                  recent                review              but          I         will  just              D is inconsistent                                 (Marsh,                unpublished                        data).
mention                some              of         the          highlights              in     the         studies                        It         is        not          possible                to     decide,               at         this       time,
on gonadal                   tissues.                                                                                            which                of the possible    mechanisms                                             of action      of
        Hall        and    Eik-Nes     (1962)                                    were      the             first          to     cyclic               AMP    on steroidogenesis                                              is the    correct
show            that    protein    synthesis                                   inhibitors,                 such           as     one.           There                  is experimental         data    in support     of
puromycin                     and     chloramphenicol,                                   blocked                     the         each.           None                  of them     is mutually      exclusive     and it
stimulatory                    effect     of LU on                              steroidogenesis                        in        may be that LU and cyclic           AMP have pleiotrop-
incubating        slices                       of rabbit                 testes.   Several     groups                            ic types     of effects stimulating    many     aspects    of
have     confirmed                            this effect                  of protein      synthesis                             the   steroidogenic     pathway.      One   part      of the
inhibitors,                  using            predominantly                            puromycin                     and         pathway                        may          be          the      limiting                step      before                   the
cycloheximide,        in testes     (Shin       and Sato,                                                     1971;              stimulation                             begins,                but      once               steroidogenesis
Moyle     et al., 1971),    corpora       lutea     (Savard                                                   et a!.,            starts              to          accelerate                    other         parts          might                   become
1965;      Marsh,     1968;      Hermier          et al.,                                                    1971),              limiting.                    A        concerted                  action             of cyclic      AMP at
Graafian      follicles        (Tsafrini    et al.,    1973)     and                                                             several                  sites         may be                 necessary              to accelerate     the
whole    ovaries        (Gorski        and Padnos,     1966).    The                                                             whole                process.
site of action        appears        to be after   the formation
                                                                                                                                                                                   CONCLUSI                 ONS
of       cyclic              AMP               in         that          Marsh            et        al.      (1966)
showed                that          puromycin                        did        not      block              the      LH               There                       is a large                     body            of evidence                           which
stimulation                      of           cyclic    AMP      accumulation                                         in         indicates                       that  cyclic                     AMP            plays  a role                        in the
incubating                   slices           of bovine     corpora     lutea,                                    but it         stimulation                      of gonadal         steroidogenesis.         This   is
did block     the stimulatory       effect                                                of       exogenous                     particularly                      true     in the         case     of the    corpus
cyclic    AMP    on steroidogenesis                                                       in        this   same                  luteum.                    There       is considerable          doubt,    however,
tissue          (Marsh              and         Savard,                 1 966a).                                                 that           it        mediates                 the effect   of small     concentra-
        Uermier               et        al.     (1971)               pinpointed                    the       site         of     tions               of     tropic               hormones     on steroidogenesis                                                  in
action            of these      inhibitors        even                                   further,               when             the        testis                or       the          adrenal             cortex.               It         is possible
they           showed      that     cycloheximide                                          acted               before             that          the              inability                of       investigators                        to       detect                a
the        cholesterol                          side-chain                  cleavage               reaction.                     change                     in         the          concentration                           of          this           cyclic
They        incubated                         slices    of           luteinized                rat ovary                  in      nucleotide                           when             small             amounts                 of          hormones
the absence                      of           02     for   90                  mm      with   LH and                             are       used,                 is due            to      methodological                              deficiencies,
found    that                   no            progesterone                         was synthesized,                               but a final      answer      to this                                      problem                    will         have          to
presumably                      due         to the lack     of 02 needed                                             for          await    further    results.
the side-chain                          cleavage   reaction    steps. When                                           the                In regard    to the possible                                              modes                of action                  of
slices    of luteinized      ovary                                        were          then    incubated                         cyclic             AMP               on steroidogenesis,                                there              is evidence
in the       presence      of 02                                           there            was     a rapid                       for an effect                           of the cyclic                       nucleotide                    at several
synthesis       of progesterone                                              and          this    synthesis                       steps in the                          steroidogenic                        pathway,                    and it may
could           not          be inhibited                          by      cycloheximide.                           This          be      that             cyclic             AMP              normally              accelerates                      several
indicated                that           cycloheximide                           did      not        act        on     the         aspects                  of      the        steroidogenic                       process                rather            than
side-chain                   cleavage                     step       or        beyond              that           point           just      one             limiting               step.
on       steroidogenesis,                             since          if it did,           it would                  have
blocked                progesterone                              synthesis               in        the      second                                                         ACKNOWLEDGMENT
incubation.                                                                                                                               I would                  like       to        acknowledge                  the     collaboration                        of
        The       role             of         RNA           synthesis                  in the            action           of      several             colleagues    who worked   with   me on the studies
LI-I       is     uncertain.                         Savard               et     al.     (1965),                  using           of the             role of cyclic   AMP in the bovine   corpus  luteum,
48                                                                                                MARSH



namely        Dr. Kenneth       Savard,       Dr.  Steven   Goldstein,                                         metabolism          in the adrenal         cortex:                                                studies on the
Dr.     Marc     G. Caron,     Dr.     Kirpal     S. Sidhu     and     Dr.                                     mode      of action      of ACTH.        Ann. of                                                the N. Y. Acad.
William      V. Ling.  I would     also like to thank      Mrs. Nieves                                         Sci. 212, 361-377.
Cerver     for the preparation       of this manuscript.                                                  Braun,     T. and Spensenwol,              S. (1974).                                                  Stimulation                of
                                                                                                               ‘AC-cyclic        AMP      accumulation          by                                             FSH and LH                   in
                                                                                                              testis    from     mature      and immature                                                    rats. Endocrinol-
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