Immunoreactive corticotropin-releasing
hormone and its binding sites in the rat ovary.
G Mastorakos, … , T C Friedman, G P Chrousos
J Clin Invest. 1993;92(2):961-968. https://doi.org/10.1172/JCI116672.
Corticotropin-releasing hormone (CRH), the principal neuropeptide regulator of pituitary ACTH secretion, is also produced at peripheral inflammatory sites, where it acts as a proinflammatory cytokine, and by the Leydig cell of the testis, where it exerts autocrine inhibition of testosterone biosynthesis. Because key ovarian functions, such as ovulation and luteolysis, represent aseptic inflammatory responses, and because the theca cell is the functional equivalent of the Leydig cell, we explored the CRH presence in the ovary, first, by specific CRH immunohistochemistry of adult cycling female Sprague-Dawley rat ovaries. We detected cytoplasmic immunoreactive CRH (IrCRH) in theca and stromal cells and in cells within the corpora lutea, at all phases of the estrous cycle. Using a specific
radioimmunoassay, we measured IrCRH in extracts of rat ovaries (0.042-0.126 pmol/g wet tissue). The mobility of the ovarian IrCRH molecule was similar to that of rat/human CRH by reverse phase HPLC. To investigate the CRH action in the ovary, we identified,
characterized, and localized CRH receptors in the rat ovary. Binding was linear with increasing tissue concentration, saturable, and of high affinity. Scatchard analysis of 125I-Tyr-ovine CRH competitive displacement curves indicated a high affinity binding site with a Kd of approximately 6 nM and a Bmax value of approximately 61 fM/mg protein.
Autoradiographic studies revealed CRH receptors primarily in ovarian theca and stroma. We conclude […]
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Immunoreactive
Corticotropin-releasing
Hormone
and Its Binding Sites in the Rat
Ovary
George Mastorakos,* Elizabeth L.Webster,* Theodore C.Friedman,*andGeorgeP. Chrousos*
*Developmental EndocrinologyBranch and the$LaboratoryofDevelopmentalNeurobiology, NationalInstituteofChild Health and
HumanDevelopment,National InstitutesofHealth, Bethesda, Maryland 20892
Abstract
Corticotropin-releasinghormone(CRH),theprincipal neuro-peptide regulator of pituitary AC17Hsecretion,is also produced
atperipheralinflammatorysites, where itacts as a proinflam-matorycytokine, andbytheLeydigcellof the testis, where it
exerts autocrine inhibition oftestosterone biosynthesis. Be-causekey ovarianfunctions, such asovulationandluteolysis, represent aseptic inflammatory responses, and because the
thecacell is the functional equivalent of the Leydig cell, we
exploredtheCRHpresencein theovary,first,byspecific CRH
immunohistochemistry of adult cycling female
Sprague-Daw-ley ratovaries.Wedetected cytoplasmic immunoreactive CRH (IrCRH) in theca and stromal cells and in cells within the
corporalutea,atallphases ofthe estrouscycle.Usingaspecific radioimmunoassay,we measuredIrCRHinextractsofrat ova-ries (0.0424.126 pmol/g wet tissue). The mobility of the
ovarian IrCRH molecule was similar to that of rat/human CRH byreversephase HPLC.Toinvestigate the CRH action
in theovary,we identified, characterized,and localizedCRH
receptors in therat ovary. Bindingwaslinearwithincreasing tissue concentration, saturable,andof high affinity. Scatchard
analysis of
'251-Tyr-ovine
CRH competitive displacement curves indicated ahigh
affinity binding
site with aKdof 6nM and a
B.
value of 61fM/mg
protein. Autoradio-graphic studies revealed CRH receptors primarily in ovariantheca and stroma. Weconclude that IrCRHandCRH
recep-tors are present in ratovaries, suggesting that this neuropep-tidemayplayaregulatoryrole inthisgonad,perhaps through its proinflammatory properties and/orbyparticipatinginthe
auto/paracrine regulation of steroid biosynthesis. Functional studiesarenecessarytodefinetherole(s)ofCRHintheovary.
(J.Clin. Invest. 1993.92:961-968.)Key words: ovary * inflam-mation*corticotropin-releasinghormone*follicle*corpus
lu-teum
Introduction
Corticotropin-releasing hormone (CRH),' a 41-amino acid
neuropeptide, wasoriginallyisolated in the hypothalamus and namedfor its propertytostimulateanteriorpituitary secretion
Address correspondence to Dr. George Mastorakos, National Institutes of Health, Building10,Room ION244, 9000 Rockville Pike, Bethesda,
MD20892.
Receivedfor publication 31December 1992 and inrevisedform16 March1993.
1.Abbreviationsused in thispaper:CRH,corticotropin-releasing hor-mone;IrCRH, immunoreactive CRH; r/hCRH, rat/human CRH;
oCRH,ovineCRH;TFA,trifluoroacetic acid. TheJournalof ClinicalInvestigation,Inc. Volume92, August 1993,96 1-968
ofACTH
( 1-3).
CRH and its receptors were subsequentlyidentifiedinmanyextrahypothalamicsites of the central
ner-voussystem
(4-7),
aswellasin severalperipheral tissues,in-cluding
activeinflammatory
sites (8-10)and thetestes (11-14).
In the centralnervoussystem, CRHplaysamajorcoor-dinativerole for the stressresponse,includingactivationofthe arousal andsympathetic systemsand elicitation of stress-re-lated behaviors(15-17).Ininflammatory sites,CRH
partici-pates as an autocrine/paracrine proinflammatory regulator
(8). Although
the actualimmune/inflammatorycelltypethatsecretesCRH is notknown, it is the immuneaccessorycells
(macrophages, tissue fibroblasts, and endothelial cells) that containimmunoreactiveCRH(IrCRH) (8-10).Inthe rat
tes-tis, CRHisproduced by Leydig cells andappearstoplayan
autocrineinhibitoryroleontestosterone secretion( 13, 14). Ovulation, luteolysis, and, perhaps,follicular atresia,three
keyovarianfunctions, havecharacteristicsofan
aseptic/im-muneinflammatoryreaction(18).Inaddition,resident
macro-phagesconstituteamajorcellular component oftheinterstitial
ovarian compartment(19-21)andmacrophage-generated
cy-tokines,suchasinterleukin 1, interleukin 6,andTNFa,have
been demonstratedorsuggestedtoparticipatein several
ovar-ian functions, suchasovulation, luteinization, and
steroido-genesis(22, 23).The theca cell in theovary,onthe other
hand,
hasbeen proposedasthefunctional equivalentoftheLeydig cellin the testis(24).The roles of CRH ininflammationand
Leydig
cell function suggested that CRH and its receptors mightalso bepresentin therat ovaryandmightparticipate ininflammatory-like phenomena and/or steroidogenesistaking placein the femalegonad.In thisstudyweidentified, quanti-tated, and localized CRH and itsreceptors in the ovaries of
maturefemale Sprague-Dawleyrats.
Methods
Animals
Adult(2-3 mo old), cycling, femaleSprague-Dawleyrats were
ob-tainedfromHarlan-Sprague-Dawley (Indianapolis, IN).Animals were maintainedon a12-h light (lights on at 0600 h), 12-h dark cycle, with food andwateravailable ad lib. Normal cycling was verified bydaily
vaginal smears. Animals were killed by CO2 inhalation, after at least three normal cycles.
Materials
Synthetic rat/human(r/h)CRH 141 was obtained from Peninsula Laboratories Inc. (Belmont, CA) (25, 26); HPLC purified '251-r/
hCRH and '25I-Tyr-ovine(o)CRH from New England Nuclear Co. (Boston, MA); cyanogenbromide-activated Sepharose 4B and 3,3-diaminobenzidine tetra-hydrochloride from Sigma Chemical Co.(St.
Louis, MO); 10% formalin (Formalde-Fresh) from Fischer Scientific Co. (Pittsburgh, PA); Vectastain ABC kits from Vector Laboratories Inc. (Burlingame, CA); rabbit IgG from Jackson Immunoresearch Lab-oratories Inc. (West Grove, PA); light green SF from RobozSurgical
(ODS-sil-ica) cartridges (C-18 Sep-Pak) from Waters Associates (Marlboro, MA); BCAproteinassay kitsfromPierce Chemical Co.(Rockford,
IL); tritium-sensitive Ultrofilms from LKB (Rockville, MD). The HPLC LKB 2150 system used was purchased fromPharmaciaLKB (Piscataway, NJ) and wascoupledto a HiPore RP-3 18 column(C-18)
(5 x 250mm) obtainedfrom Bio-RadCo.(Richmond,CA).
Tissuepreparation
The ovaries were dissected immediately after killing. Ovaries prepared for immunohistochemistry were fixed in 10% formalin and embedded inparaffin.Ovaries prepared for CRH extraction and radioimmunoas-say, CRH bindingassay, or autoradiographic mapping of '251-Tyr-oCRH-bindingsites, were frozen on dry ice immediately and stored at -70'C until assay.
Immunohistochemistry
Peptide antisera production andpurification. Polyclonal antiserum
(TS-2) against r/hCRH was produced in rabbits as previously
de-scribed (27). Beforeuseit was affinity-purified by adsorption to
syn-thetic r/hCRH 1-41 coupled to cyanogen bromide-activated Sepha-rose 4B.Briefly,the antiserum was added to the r/hCRH-Sepharose 4B
conjugate( 1 mg of r/hCRH 1-41 on 200Mggel)and incubated at room temperature for 2 h. The suspension was packed in a 4 x 0.7-cm col-umnand washedwith phosphate buffer (20 mM sodium phosphate, 0.5 Msodiumchloride,pH 7.3) until theOD2W returned tobaseline. The elutions obtained fromthese washings were depleted ofanti-r/
hCRH IgG(affinity-negativeIgG fraction) and were used in
immuno-histochemistry staining as controls ofthe specificity of the anti-r/
hCRH IgG. Theantibodybound to theaffinity-column
(affinity-posi-tive IgG fraction)waselutedwiththiocyanate buffer (3Mpotassium thiocyanate, 0.5 M ammoniumhydroxide)anddialyzed against sev-eralchangesofcoldPBS.
Immunohistochemistry. Tissue specimens were preserved in 10%
formalin. They were embedded inparaffinandsectioned onto gelatin-coatedmicroscope slides at a thicknessof6gm.Immunoperoxidase
stainingwasperformed withtheVectastainABCkit (Vector
Laborato-ries), usingthe manufacturer's suggestedprotocoland reagents(28,
29).Allsubsequentprocedurestookplaceat roomtemperature. The
sectionsweredeparaffinizedwithtwo5-min washes inxyleneand
rehy-dratedby sequentialrinses inabsolute,90%, 80%, and70% ethanol.
Endogenous peroxidase activity wasexhausted by incubation with 0.3%peroxideinmethanol for45 min. Thesectionswerethen incu-batedsequentiallywith0.1%BSAinPBS for20minandwith diluted
goat serum (1/66.7) for20min, and inahumidchamberwiththe
affinity-purified rabbit antibodytor/hCRH(50Mg/ml),thecontrol,
affinity-negativeIgG fraction(50Mg/ml)ornonimmune normal
rab-bit IgG (50,g/ml).After40min,thesectionswerewashedwithPBS andincubated withbiotinylatedgoatanti-rabbit IgG for30 min. The
sectionswerefurtherwashed with PBS and incubated with avidin anda
biotinylated horseradishperoxidasecomplexfor 45 min.Finally,the
sectionswerewashed and colorwasdeveloped by immersingsections in asolutionof 0.05%wt/vol3,3-diaminobenzidine
tetra-hydrochlo-ride,0.04%wt/vol nickelchloride, and 0.01%hydrogenperoxidein 0.05MTris,pH 7.4, for 2 min. The sectionswerecounterstained with 0.5%lightgreen SF. Positivestainingwasrevealedasblack-dark green spots, whereaslightgreen colorwas seeninr/hCRH-freeareas,
affin-ity-negativecontrolsections,and nonimmune normal rabbitIgG
con-trolsections.AdifferentstainingwasalsoobtainedbyusingtheDAB
solution without nickelchloride,andMeyer's hematoxylineas nonspe-cificcounterstainingof cell nuclei. Inthis case,positivestainingwas
revealedasbrowncolor, andnonspecific stainingof all cellnucleias
lightblue color.
Radioimmunoassay
Ovarianconcentrations of CRH were measured by radioimmunoassay after acid extraction. Briefly, 10 volofboiling 2 M acetic acid was
addedtowhole ovaries and the mixturewasincubated at 950C for 10
min.SampleswerethensonicatedoniceforImin andcentrifugedat 15,000 g for 30 min. 3 vol of acetonewasadded to the supernatants, andtheywereagain centrifugedat15,000gfor30 min. The
superna-tantswerecollected,lyophilized,andreconstitutedin radioimmunoas-saybuffer formeasurement,aspreviously described (30).TheCRH
antiserum (TS-2) used has been characterized in detail previously (27). 1251-r/hCRHwasusedasthe tracer, andsynthetic r/hCRHas
the standard. Thewithin-assay coefficient of variationandsensitivity
were 4% and 1.05pmol/liter, respectively.
Reverse
phase
HPLCThesamples obtainedfromacid-extractionof the ratovarieswere re-constituted in 200Mlof0.1%trifluoroaceticacid (TFA). The sample
was then passed through an ODS-silica cartridge (C-1 8 Sep-Pak; WatersAssociates),and thecartridgewaswashedwith0.1%TFA, and
subsequently by20%acetonitrilecontaining0.1% TFA. Thepeptides
werethenelutedoffthecartridgewith 5 mlof60%acetonitrile
contain-ing0.1% TFA. The eluatewaslyophilized, reconstitutedin 200Mlof
0.1%TFA,andanalyzed byHPLC. Analiquot of 180Mlofthe sample
wasinjected intothe HPLC column system.Solvent A was 0.1%TFA and solvent B was 80% acetonitrilecontaining 0.1% TFA. A linear gradient, from40to65%ofsolvent Bat aflowrateof1ml/min,was
appliedover45 min. 1-mlsamples ofthe eluatewerecollected inan
automaticcollector,lyophilizedtodryness,reconstituted with radioim-munoassaybuffer,andassayedfor CRH content,asdescribed above.A
standard of 1Mgofsynthetic r/hCRHwasinjectedafter each
experi-ment todetermineitschromatographyprofile.
CRH
binding
assay
Theaffinityandconcentration ofCRHbindingsitesweredetermined from competitive displacement binding experiments, in which rat
ovarian tissue homogenatesand'251-Tyr-oCRHwereincubated with increasing concentrations ofunlabeledsynthetic r/hCRH. Both
ova-riesofthesameanimalwereusedtoproduceeachdisplacementcurve. Atthe timeofthe assay ovarieswereweighedandsubsequently homog-enized in 20 volofice-coldhomogenization buffer (50 mMTris-HCI,
10 mMMgCl2,and 2 mMEGTA, pH 7.2) usingaPolytron
(Brink-mannInstruments,Westbury, NY).Thehomogenatewasthen centri-fugedat38,000g for 10 min, the supernatantswerediscarded,and the
pelletswereresuspendedto afinal concentration of40 mg tissuewet
wt/ml inhomogenizationbuffer. 100 Mlofthe membranepreparation
werethenaddedto a 1.5-mlpolypropylene microtubecontaining 100 Ml ofanr/hCRHsolution(12 concentrationsrangingfrom 0to1MM)
and 100Mlof
'251I-Tyr-oCRH
(0.2nMfinalconcentration)inincuba-tion buffer(50 mMTris-HCl, 10 mM MgCl2, 2 mMEGTA, 0.15%
BSA, 100kallikrein inhibitorunits/mlaprotinin,0.15 mMbacitracin,
pH7.2).Thereactionwasallowedtoproceed,at roomtemperature, for 2 h. The membraneswerethenseparatedfrom the incubation
me-diumby centrifugation in a microfuge at 12,000g for 8 min. The supernatantwasaspirated,thepelletswashedgentlywith 1 mlofPBS
containing0.0 1% TritonX-100,and theradioactivityof thepelletwas
measured ina y counter.Thefinalproteinconcentrationwas deter-minedusingtheBCAproteinassay.
Autoradiographic
mapping
of'25I-Tyr-oCRH-binding
sitesSlide-mounted tissue sections from ovarieswereincubatedatroom
temperature with 0.1nM
'23I-Tyr-oCRH
inincubation buffer(50mMTris-HCl, 10mM MgCl2, 2 mMEGTA,0.15%BSA, 100 kallikrein inhibitorunits/mlaprotinin,0.15 mMbacitracin, pH 7.2)for 2 h.To
assessnonspecificbinding parallelsectionswerealso incubated in the
samesolution of 25I-Tyr-oCRH in the presence ofI MM r/hCRH.
Afterincubation,tissue sectionswerewashed in PBScontaining0.0 1% Triton X-100at40Cfortwoconsecutive 5-minperiods,dippedina
deionizedwaterrinse,anddriedrapidly,underastreamofcolddryair.
Tritium-sensitive Ultrofilmwasapposedtothetissue sections. After
4-5d ofexposure,the
autoradiograms
weredeveloped,and the tissueI
0
C
7)
9
0
"'A~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~J
* **
2 ;j$** ':.t.
* 88_ _ ; ;s * < x * 8 i..
Dataanalyses
'25I-Tyr-oCRH bindingdatawereanalyzed bytheMacintosh-based
nonlinear least squarescurvefittingcomputer programLIGAND(31).
Results
Immunohistochemical detection ofIrCRH in the rat ovary. The presence of IrCRH in the ovaries of adult, cycling Sprague-Dawleyratswasexamined byimmunohistochemistry
of sections obtained at proestrous, estrous, metestrous, and diestrous. IrCRHwasdetected in thecytoplasmof theca and stromalcells,regardless of sizeormaturity of ovarian follicles (Fig. 1, A and C),aswellasin the oocytesofantral follicles (Fig. 2 C)butnottheprimaryoocytesofprimordialfollicles (datanotshown). Asmallamountof CRH immunostaining
was also seen in some granulosa and surface epithelial cells
(Figs. 1 C and 2 C).IrCRHwasalso detected in the cytoplasm ofasubpopulationof cells within thecorporalutea(Fig. 1, D andF,and 2A).These cellswerealsofoundthroughallstages
ofcorpora lutea maturation and at all phases of the estrous
cycle.
Since the 14-dhalf-life ofarat corpusluteumislonger
thanthedurationof the 4-5-drat estrouscycle,corpora lutea of different maturationstages werefound in theratovariesatallphases of theestrouscycle. TheIrCRH-containingcellsof thecorporalutea, however, werelocalizedprogressivelyfrom the peripheryto the center with advancing maturation and therewaslessstainingfor IrCRH in oldercorporalutea
com-paredto younger. Thematuration/aging ofthecorporalutea
was based on morphological criteria, including the
progres-sivelyincreasingpresenceof fibrotic tissue withadvancingage
of thecorpusluteum.
IrCRHmeasurements and HPLC mobility in rat ovarian
extracts. Total ovarian IrCRH content wasmeasured in ex-tracts ofrat ovaries at all phases ofthe estrous cycle. The
amountvaried between 0.042 and 0.126 pmol/g ofwettissue. No significant differences in the CRH content were found
among ovaries obtained atthedifferent phases ofthe cycle.
Ovarian IrCRH hadsimilar chromatographic mobility to r/ hCRH 1-41, theformproduced by theratandhuman hypo-thalamus and the human placenta (Fig. 3). Therewerealso
immunoreactive bands with higher mobility than r/hCRH 1-41, indicating the presence of CRH-degradation products and/or aggregates/precursors with different hydrophobicity.
Toexamine whethertheovarianCRH levelsreflectedorwere
reflectedbycirculatingCRH levels,we measured plasma
con-centrations of IrCRH in the same animals. Plasmalevels of IrCRH remained <2.101 pmol/liter (10-2 M) throughout theestrouscycle.
CRH
binding
characteristicsof
rat ovaries.Specificbinding
of
'25I-Tyr-oCRH
wasmeasured inhomogenatesofratovariesusingassay conditions identicalto thosedescribed for
mem-branepreparations from mouse pituitariesand spleens (32). Specificbindingof radiolabeledoCRH was linear with
increas-ingtissue concentrations. Competitivedisplacement curves of
125I-Tyr-oCRH
binding in ovarian homogenates incubated withincreasing concentrationsofr/hCRHdemonstratedthat thespecific bindingwas saturable (Fig. 4).Scatchardanalysesusingthenonlinearcurve fittingprogram LIGAND (31 ), re-vealed a single, high affinity binding site (Fig. 4, inset). The apparent Kdofovarian CRH binding sites was 6.34±2.3 nM
(mean±SEM,n = 13) and the receptorconcentration
(B.,,.,)
was61.17±15.6fmol/mg protein.
a 0 CO_o 0 E I CD 0 -q u
.-0 10 20 30 40
Fraction number
Figure3. ReverseHPLCcharacterizationof IrCRH extractedfrom
ratovaries. Theamountof CRH in individual fractions was
deter-mined byRIA.Synthetic r/hCRH 1-41 elutedfromthiscolumnin
thefractionindicated byarrow.
Autoradiographic distribution of '25I-Tyr-oCRH-binding
sitesintheratovary. Theregional distribution of CRH
recep-torswithin therat ovary wasinvestigatedusing in vitro
autora-diographic techniques. Autoradiograms on tritium-sensitive Ultrofilm (Fig. 5B) werecomparedtophotomicrographs of
thesametissue section stained with thionin (Fig. 5 A). CRH
receptors were diffusely distributed in ovarian theca and
stroma areassurrounding follicles aswell as onthe cumulus oophorus (Fig. 5 B). Therewas anotable absence of specific binding in the granulosa areaswhile specific 125I-Tyr-oCRH binding sites in thecorporaluteawere sparseandnot
conclu-125I.oCRH
BINDING IN
RAT
OVARY
0.022
0.00,7S 0
0.020
0o.oo2s
10 20 30 40 50 60
c Bon(fto"pi
0
m 0.018
0
0 .1 1 10 100 1000 CRH Concentration(M)
Figure 4.Competitivedisplacementbinding of'251-Tyr-oCRH
dem-onstrating specific bindingsites inratovarianhomogenates incubated in the presence ofincreasingconcentrations of r/hCRH. Competitive displacementbindingof'25I-Tyr-oCRHdemonstrates total binding
as afunction of increasing ligand concentration. The Scatchard plot (seeinset)representsspecificbindingdata (totalnonspecific
bind-ing). Scatchard analysis of thedisplacementcurves by the program LIGAND(31) revealedasinglehigh affinity binding site. The data shownarefromarepresentative experiment. The dissociation con-stantof6.34±2.3nMand the
B.,,
of 61.17±15.6fmol/mgprotein (mean±SEM) were determined from the results of 13 independent0%
of
.I-4IA i
r* ...g~~~~~A ,.
.
.. z w
.C
,
I"
1//
~~th
pf
g
at
B~~~~~~~~~
i '
cl~~~c
A~h
Figure5.Autoradiographiclocalization of CRH receptors in therat ovary. (A)
Bright-field photomicrograph showingthe
histologyof the section ofrat ovaryused
togeneratethe autoradiogram shown in B.
AfterexposuretoUltrofilmasdescribed inMethods,thetissuewasstained with
C,^
thionin.(B)Directprintoftheautoradio-gramgeneratedontritium-sensitive Ultro-film, whichshows thedistribution of
auto-radiographicgrains(whitespots)in the section of ovary shown inA.(C)
Nonspe-cificbindingdetermined in thepresence
ofI mMr/hCRHwasuniform.Proestrous
ovary sectionsareshown here. Ovariesat
allphasesof theestrouscyclewere
exam-ined,however. s, stroma; g,granulosa;af,
atreticfollicle; cl,corpusluteum;co,
cu-mulusoophorus;th, theca;pf,primordial
sivelyseen. Bindinginthepresenceof1
gM
r/hCRHwaslow anduniformly distributed (Fig. 5 C).Discussion
We havedemonstratedthatIrCRHislocalizedin the normal
rat ovary.Ovarian IrCRH hassimilar chromatographic mobil-itytothat of r/hCRH 1-41, the form produced by therat and
human hypothalamus, rat and human peripheral
inflamma-torysites,ratLeydig cells,and humanplacenta(30,33,26,34, 8, 9, 10, 13, 35). The concentrations of IrCRH in theratovary (0.042-0.126 pmol/g ofwettissue)aresimilartothose found in the extracts from rat inflammatory tissues (0.084-0.105
pmol/gofwettissue)(8,9)orhumanarthriticjoints(10), but
arelower than those in therattestis (10.719-15.554 pmol/gof
wettissue)( 11).Ovarian CRH could be derivedfromseveral
sources.First, it might be producedbyimmune accessory cells normally residing in the ovary, suchas macrophages, tissue
fibroblasts, and endothelial cells (19-21). CRH mRNA and
peptide have been shown in peripheral blood mononuclear cells (36) and in spleen and inflammatory synovia from
ar-thritic Lewisrats(9), whereasthe presenceofalarge number of extravascular macrophages has been well established in the ovarianstromaandcorporalutea(19-21, 37). These
macro-phagesconstituteamajor cellularcomponentof the interstitial ovariancompartmentandaremostly concentratednear
perifol-licularcapillaries ( 19-21 ). Second, another potentialsourceof ovarian CRHmightbetheterminalsof postganglionic
sympa-thetic and/orunmyelinated sensory neurons type C and Ab. IrCRH ispresent atlarge concentrations in both the
sympa-theticchainandthe dorsalrootganglia,where the cellbodies of theseneuronsreside(38, 39)andis depleted in theratspinal cord anddorsalrootgangliainresponse tocapsaicin,whichis toxicto sensoryafferent fibers(40). Third,we mustconsider
thattheca andstromacells, luteinized cells in thecorporalutea, andmatureoocytes, all ofwhich containedIrCRH,might also themselves produce this peptide. The plasma is an unlikely
source of ovarian CRH, since the levels of this peptide inrat
circulationareextremely low(8).
Inthepresentstudy,wealsoidentifiedspecific,high affinity CRHreceptorsin homogenates ofratovaries. '25I-Tyr-oCRH binding wassaturable and increased linearly with increasing protein concentration. Scatchard analysis of competitive
dis-placementcurvesrevealedahighaffinityreceptor for CRH in rat ovary (Kd = 6.34 nM), correspondingto the highaffinity
receptorpreviously identified in ratpituitary, hypothalamus, brain (6, 7), Leydig cells( 14) andarthriticsynovialtissues(9),
andmousespleen (32).Thebinding capacity ofthe ratovary homogenates
(Bm.
=61.17fmol/mg protein)wasin the same order of magnitude as that ofrat brain cortex homogenates(Bmax
= 62.19 fmol/mg protein) (32, 6), one-tenth of that shown inpituitarygland (41, 42), and 6 times and 20 timeshigherthan that shown in mouse spleen
(Bm.
=8.74fmol/mg protein) (32) and in rat Leydig cells(Bm.
= 2.6 fmol/mg protein) (14), respectively. We examined the distribution of'25I-Tyr-oCRH
binding sites by in vitroautoradiographytode-termine whetherCRHreceptorsareexpressedbyspecific cell typeswithinthe ovary. Thereceptorswereprimarilyfound in stroma and theca cellssurroundingfollicles,regionsknown to
contain macrophagesand otherimmune accessorycells ( 19-21),but alsointhe cumulusoophorus, which might indicate
interactions between the IrCRH-containing mature oocyte and cells of the cumulus. Thereweresparse, dispersedCRH
receptorsin thecorporalutea,whichmightalsobeattributedto
presenceof residentimmune accessorycells in theseareas(20, 21, 37).Neither in thetheca/stromanorin thecorporalutea
can weexclude thepresenceofreceptorsfor CRH on the actual
steroidogenic cells,however.
The presence ofCRH and its receptors in the rat ovary
raises thequestionoflocalinvolvementofthispeptidein
ovar-ian physiology. Twopotentialrolescanbeenvisioned by
ex-trapolation from existing information. First, ovarian CRH
mightactas aproinflammatory cytokine duringthe inflamma-toryphenomenaofthe ovarianphysiology,which include
ovu-lation, luteolysis, and, perhaps, follicularatresia. Theca CRH
might participateinthe actualprocessof ovulation and in the
vascularizationofthegranulosa during formationofthecorpus
luteum,wheneosinophilsand Tlymphocytes chemoattracted
into the follicleproducelymphokinesthat attract and activate
monocytes/macrophages (43). During luteolysis, phagocyti-callyactivemacrophagesappeartobe involved indigestionof
lutealcells( 19, 44).Thegeneral presenceofCRH inperipheral
blood mononuclearleucocytes (36) and other inflammatory
cells, anditsproinflammatory properties (8-10) suggestthat thepeptideofthecorporaluteamightbeinvolvedin the im-muneaspectsofluteolysis.OvarianCRHmightexertits
inflam-matoryactionsdirectly and/or indirectly via cytokines, since
manyofthe effects of this neuropeptideon peripheral blood mononuclear cells aremediated by IL-1f (45). The second rolethatmight beascribedtoovarian CRH is its participation
inovariansteroid hormonebiosynthesis,inanalogytoits effect ontestosterone biosynthesis by Leydigcells ( 13, 14).Again,
thiseffect might be direct or mediated by local secretion of
cytokines and/or lipid mediatorsofinflammation, knownto
influence hormonogenesis. Indeed, IL-1 mediates inhibitionof
hCG-stimulated androsterone accumulation by cultured wholeratovarian dispersates and might, thus, be involvedin the actions of CRH(46). Further studiesareneededto define thebiological role(s)ofovarianCRH.
Acknowledgments
E. L.Websterissupported by the pharmacology research associate
program(PRATfellowship) oftheNationalInstitute ofGeneral Medi-calSciences.
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