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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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(2)

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 a

high

affinity binding

site with aKdof 6

nM and a

B.

value of 61

fM/mg

protein. Autoradio-graphic studies revealed CRH receptors primarily in ovarian

theca 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 subsequently

identifiedinmanyextrahypothalamicsites of the central

ner-voussystem

(4-7),

aswellasin severalperipheral tissues,

in-cluding

active

inflammatory

sites (8-10)and thetestes (

11-14).

In the centralnervoussystem, CRHplaysamajor

coor-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/inflammatorycelltypethat

secretesCRH 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 in

inflammatory-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

(3)

(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

HPLC

Thesamples 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)in

incuba-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

sites

Slide-mounted tissue sections from ovarieswereincubatedatroom

temperature with 0.1nM

'23I-Tyr-oCRH

inincubation buffer(50mM

Tris-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 tissue

(4)

I

(5)

0

C

7)

9

0

"'A~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~J

* **

2 ;j$** ':.t.

* 88_ _ ; ;s * < x * 8 i..

(6)

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 corpusluteumis

longer

thanthedurationof the 4-5-drat estrouscycle,corpora lutea of different maturationstages werefound in theratovariesat

allphases 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

characteristics

of

rat ovaries.Specific

binding

of

'25I-Tyr-oCRH

wasmeasured inhomogenatesofratovaries

usingassay 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).Scatchardanalyses

usingthenonlinearcurve 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 independent

(7)

0%

of

.I-4I

A 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)Directprintofthe

autoradio-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

(8)

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 times

higherthan 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 vitroautoradiographyto

de-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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