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INFORMATION TO USERS

This manuscript hasbeenrepoduc:edfromthemicrofilmmaster. UMINms thete xtdire<:tlyfromh originalOfCDf1f~, ThJs.some ....sis and dissertationcoPiesareintypewriterface.whileotherSmaybefromanytypeof comput er printef.

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Ontogenyof the Androgen Receptor in the Hippocampus ofthe Sprague-Dawley Rat.

BY

DorisM.Babstock,B.Sc.

A

thesis

submitted tothe School01GraduateStud ies

in partialfulfillm entof the requirements for the degreeof

Doclor ofPhilosophy

Departmentof Psychology Memorial Universityof Newfoundland

December1999

St. John's Newfoundland

(10)

Abstract

hlYl'lJnocytoc:hen'lstry (ICC)with a P<>'ydonal antibody (PG·21)was used 10 inve stiga tetheontogenyofthe androgenreceptor(AR)in the hippocampus of Sp raque -Dawieyrats.Brain s of fivemale andfemaleHttermateswereexamined at each agecove ring theneonatal(PND2,5.7),juvenil e (PNO14),prepubertal (PNO 25),andpubertal(PNO40)periodwith one maleand femaleexarnnedat PND60.Aswelt.twosets ofPNO 7andPND 25 maleandfemale lirtermates wereinjected 30 minutes beforeperlusionwithdihydr ot est oster onebenzoate (D HTBllolook fora possible ligand-dependenlincr easein stain ing .The density ofsta iningin the hippocarrpalCAl. CA3andOGcell layerswas determinedin each sectionwith the densityofth eDG sub tractedfrom the density al tha pyramidalcentayers as acontrol for backgroundstaining.

In the nippocarrpusofbothsexes, ARstainingden sityis describedby an inverted

u-snacee

function betweentheages ofPNO2andPNO25with a peak at PNO7.AAstaini ngdensity in theCAlcelllaye risat itshighestden sity in both sexesduringpuberty(PNO40),withsignificanUy gre a ter density inthe

male.In contrast,atPNO40. ARstainingdensity remainslow intheCA3 cell

layer.Theonly otherage thatexhibits apossiblesexdiffe renceisPND7 with a trendwithi n the CA 1cell layer forgreate r AR stain ingdensity in the male.

Intheneonatal andprepubertal animal,theARstaining isdispe rsed equallythroughout thecell withnuctearenha nced densityobserved atPND 40.

(11)

Theeffect of DHTB treatment on AR densitydepends onage.Increased staining

is observedinbothsexes atPNO7whereas,intheprepubertalanima ls(PND

25) ,DHTB appears to decrease ARdensityinfemale swithno consisten teffect inmales.

Theontogenyof theARwithinthetappccarrousis COf'Tl>aredto the ontogeny01 the ARin otherbrain areasandtothe ontogenyof the estrogen receptor (ER).Itis concluded thatneonataland prepubertal hippoc ampalAR mayberegulatedbyfacton other thancircul atingandrogen as ARdensityisnot clearly related toserum testosterone(T)levels andOHTB-treatment does not elicitanuclear transloca tion asdemo nst ratedin adult animals.Instead ,theAR

is equallydispersedthroughoutthecell,regardless of androgentreatment.

Thus,the neonatal and adultAR may bedifferently regulatedand may have different functions.

Keywords:Androgen recepto r(AR).irrmmOC'ytoc hemistry (ICC),PG-2 l, ontogeny,hippocar11)Us.CAl,CA3,dihyd rote stosteronebenzoate (DHTB).

(12)

Acknowledgements

Iwishto expressmygratitude toDrs.J.McLeanand R.Adamec who served onmycommittee.I also acknowledge andappreciatethecontributionof mysuperviso rs.Drs.C.Haney and C.Malsburyfor thei rrroraJ and technical supportthroughout the many stages of thisproject.A specialthanks toDr.

Malsbury forhis encourag ementandins ightfulcommentsas wellasfor providi ng the facilitiesthat enabled this worktobe

cororeteo .

Iwould also like tott\ankNSERCandthe SchoolofGraduateStudieslor thei r financialsupp ort during theearly stages ofmypostgraduat e studies.As well.Iwish 10expressa very special thanks toOr.GailPrins,University of Illinoisat Chicago .OrPrin s generous giftofAAantibodyandcontrol peptides ensuredthe survival andcompletion of thisproject.

Finally,very deepand heartfelt thanksgoto"'i farrily,without whose support.friendshipandlov e,Icould neverhaveconsidered striving forand possiblyrealizin g thefulfillmentof thisdream.Thank you Cindie ,Caryn&Krista and averyspecialthank-youtorrr'(husba nd and lifecoroamon.Reg inald.

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TABLE OF CONTENTS

Abstrac' 11

Ac knowledgements Iv

Lis tofFigures vi

List of Abbreviations vIII

Ontogeny of the Androgen ReceptorIn the Hippocampusof the Sprague-

DawleyRat. 1

CHAPTER 1:INTRODUCTION 1

1.1Synthesis01SteroidHormones 3

1.2OntogenyofAndrogenSecretion 4

1.3 c-Fetoproteo 7

1.4 Aromatizalion 8

1.5 Sa-Reductase &DHT 12

1.6Oefeminization&Masculinization 14

1.7FunctionalDomainsandNeuronalLoca tionofthe AndrogenRece ptor_ 25

1.8Ontogen y01theHippoca mpa l Formation. 31

1.9TheAndrog enRecep tor:withinthe Hippocamp us 35

1.10SexDillerencesand theHippocan..,us 39

CHAPTER 2:METHOD 48

2.1 Animals 48

2.2AndrogenReceptorAntibody SO

2.3~ 51

2.4 Analysis 54

CHAPTER 3:RESULTS 57

3.1DO 57

3.2~1 ~

3.3~ ~

3.4 Contro!Sections 66

3.5OHTSatPNO7 67

3.6OHTSetPNO25 73

CHAPTER4:OCSCUSSION 81

4.1Comparison01the OntogenyofHjppoca~lAAwithOtherAreaswithin the

~S ~

4.2Comparisonof the OntogenyofHippocampal AAwithEA 84

4.3AR RegulationbySerumT? 86

4.4 ConformationalChange and DHTB 93

4.5 PossibleFunctionsofthe ARwithintheHippoca~us 99

References 109

(14)

List01 Figures

Figure1.1 Drawingof thedorsal hippocarf1)US of the ratshowing both

Anvnon'shorn and thedentate gyrus 34

Figure 2.1 Computerdigitizedvideo image ofthe mpoccarrous from aPND 60male illustratingthemethOdused10sarrpleAR-fr 55 Figure3.1 Ontogen icprofile oftheaverage ARdensity:I:SEMwithin the OG

cell layer of the dorsalhippoca mpus...•..•...•... ...58 Figure 3.2 Ontogenic profileofthe averageARdensity:l:SEMwithinthe

pyramidalcell layer of CA 1 ofthe dorsal hippoca mpus 59 Figure 3.3 (A) and(B).Photomicrograph of representativesectionsshowing

theCA1.CA3andOGfrommale and female ratsat eachage

analyz ed. . 60

Figure 3.4 (A) and(8)Photomicrogr aph ofrepresentative sectionsshowing low andhigh magnifica tionof theCA1celllayerinthe PND7and PND40animal...•...•...•..•....64 Figure 3.5 Ontogenicprofileoftheaverage ARdensity1:SEMwithin the CA3

celllayer ofthedorsal hippocarTlJus...•...65 Figure3.6 Photomicrographof sections processedinthecontrol studies from

male.atPND7 (a),(b)and at PND 40 (e),(d) "",.,68 Figure3.7 Results obtainedwithinCAlfollowing trea tmentwith CHTSat PNC

7 ,,, ,, ,, ,, ,, " 69

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Figure3.B Photomicrographofrepresentativesecti o ns fromPNO7control

andOHTB·treatedmales andfemales 70

Figure 3.9 PhOtomicrographofrepresentative sectionsfromPND 7control andOHTB·trea tedmales and femalesathighmagnification

showingthehippocampal CA lcell layer 71

Figur e 3.10 Resultsobtainedwithin CA3followi ngtreatme ntwithOHTBatPNO ...72

Figure 3.11 Results obtainedwithi n OG following treatmentwith OHTBal PNO

7... . 74

Figure3.12 Resultsobtainedwithin CA1 following treatmen t withOHTBatPNO

25 . ...75

Figure3.13 Photomi crographof representativesectionsfrom PND25 control andOHTB-treated malesathigh magnificati on showing the

hippocarroalCA 1celllayer 76

Figure 3.14 Photomicrograp h ofrepresentative sectionsfromPNO25control and OHTB·lreatedfemalesaltwodifferentmagnificatio ns 77 Figure 3.15 Results obtainedwithinCA3followi ngIrea tment withOHTBatPNO

25 . .. 79

Figure 3.16 Resultsobtainedwithin OG foll owingtreatmen t with OHTBat PNO

25 80

(16)

AR21 AR462 AR BSA cAMP DG DAB DHT DHTB E EB E, ER EPSP GR

hER hAR

ICC -ir ISO LTP mRNA ORX OVX PND PR SNB s.

sc R1881

or

TP

Us!of Abbreviatio ns

Apeptide usedto raisetheantibody Adistantpeptide

androgenreceptor bovineserumalbumin cyclicadenosine3'.5'-phosphate dentategyrus

diamnobenzidinetetrahydrochloride dihydrotestosterone

dihydrotestosteronebenzoate embryonicday estradiolbenzoate estrogenIestradiol estrogenreceptor

extracellularpopulation spikepotential glucocorticoidreceptor humanERorAR

irrmunocytochemicalJimmunocytochemistry immunoreactive staining

InternationalStandardOrganization long-termpotentiation messengerRNA orchidectomy ovariectomized postnatal day progesteronereceptor

spinalnucleusofthebulbocavernosus stratum(e.g.,e.oriens) subcutaneously

synthetic non-aromatizableandrogen,methyltrienolone testosterone

testosteronepropionate

(17)

Ontog enyof the Androgen Receptor in the Hippocampusofthe Sprague-Dawl eyRat

CHAPTER t: INTRODUCnON

The brai nregulates avarietyof function s thataredifferent between males andfema les.Some ofthesedifference s.termed 'organizationaleffects'.are producedbyperina tal expos ureto thesex steroids,whichpermanentlyalters neuralconnectionsandfuncti on s withinthebrain.Othe r functionaldifferences occur throug houtlifewithtempo rary effectsupon neuralsubst rates orcircuits, thusearnin g thenomendature'act ivation aleffects'.Bothtypesresultfromthe endogenou shormo nal environmentthatdiffersbetween malesandfemal es.

AlthOugh theremaynetbeatruedichotomy betweenthese two classes(see Arnold &BreedloveG),thetermino logy remains gene rally useful andwillbeused asdefinedabove.The most obviousandbest srudiedfunct iona ldifferences between matesand females arethose invcivedinreprod uct ive physiologyand behavior.Conse que ntly,muchisnow kno wnabout the effect 01the sex steroi ds onbehavior .physiologyand neuroanatomythaiislinkedto mating(torrevi ew see'.2&43,5'.S5 ..._109.1I••1I11.1~.However,relatively recent discoveriesof the interacti on between prog es teron e andthe GABA recept or,•.n.~,lI2.!I3.l(IO,':N,l "O.l~

as wellas theeffect ofestrog en(E2)andtheestrou scycleondendritic proce sses withinthehippoca r11>US5oI.Ios.11&.'79suggeslthat the sex steroidsmay beinvolvedinmore thanreproductive behavior'~.

(18)

As a means of introducing the classical conception of the androgen receptor (AR) within thecentral nervoussystem, theintrod uction will reviewthe involvement 01androgens andtheARinreproductive behavioras defined by defeminizati on and masculiniza tion.Thiswill eocceoass such topics as the synthesis01steroid hormones,the ontogenyof androgen secre tion,the presenceand lunctionof

c-r erccrcteo.

andthe roleof aromatizationin brain development.As well,resultsfromtreatme ntwiththenon-aromatlz able androgen,dihyd rotestosterone(DHT),willbereviewed to helpdefinethe involvementofthe ARin these processes.Theintroduction willthen examinethe composition and propertiesoftheAR protein,asthis proteinis the meansof explorationutilizedbythecurrentstudy.Becausethis thesis isconcernedwith thepresence of the AR withinthe hippocampus of the neonate,theontogeny of the hippocampa lformation will thenbeintroduced aswell as whatis currently knownabout the presenceof thisreceptorwithinthisbrainarea.Final ly,the introd uctionwill revie whippocampal-rela tedsexdiffer ences to providea frameworklorfunct ions that may be relatedto the presence of the ARwithin the hippoca!T1lalformatio n.

In general,beforearecepto r caninitiate eithera singl eor a cascadeof events,itmustfirstbindtoits activatingligand.This is also true of thesteroid receptors.Thus,the first topic tobecoverediswhenand howthe activa ting ligands of the steroidrecep torsarefirst synthesized.

(19)

1.1 SYNTHESISOFSTEROID HORMONES

As reviewedbyBrown21,horrrones areclassifiedintotwodistinctgroups, steroidsand peptide/protein hormones.Thelatter are synthe sizedfrom anino acidswithin theribosomesof the roughendoplasmicreticulum".Theirfunction and descriptionwill notbeelaborated here as they donot relateto this project.

The sex hormones belongtothe steroidgroup,synthesiZedinthe srrooth endoplasmicreticulum ofthecellbodywithinthegonadsandtheadrenal cortex 21.Followingsynthes is.they aretransported totheir respective sites of action within the brain viatheendocrine system(blood stream).Steroids are synthesized 'rom plasmachofester~,which isinitiallyconvertedinto pregnenolone,andtheninto progesterone

2'.

Both ot these substances can be hydroxylated.Withinthegonadsof both sexes,hydroxyprogeste roneacts as a prohormoneto testosterone('T)and OHT,which are secreted fromthetestes21, Within theovaries,smallamounts ofT areexcreted withou tconversion but mostis further converted to estradiol (E2)21.Inthe adrenal gland,progesterone canbesecre tedas a steroid hormone or act as a prohormone(precursor) to the corticosteroids,corticosterone and aldosterone21.However,thehydroxylated form ofproges teroneactsas a prohormone tothecorticosteroid,hydrocortisone ortothe sex steroids,'T and E221.Althoughthegonads andadrenal glandare proven sourcesofthe sex steroids,recentworksuggests thatsteroidsmayalso besynthesizedwithinthe brain.earningthenomencl ature neurcstercids125.

However,althoughneuronsmetabolizegonadalsteroids(e.g.,aromatization),

(20)

thereis no evidence thatdenovosynth esis ofsexsteroid s withi ntheeNS plays aroleinitssexualdifferentiation.

1.2ONTOGEN YOFANDROGEN $ECRETJON

The perinatal periodis a criticalperiodfOf the organizing effects ofT on brain and behavior.In orderto understandthe organizingprocess itis necessary toknowa)when the brainis exposedtoTand blwhentheparticular brain region s begintoexpress steroidrecep tors.This sectionwill explore whatis knownaboutthe ontoge nyoftheligan d thatactiva testheAR.

Using radioirrmu noa ssay.'Tisfirst detectedin the testides ofthe Sprague-Dawtey33and Wista r30lmale ratbythe1""'-18'"dayofgestationandin the ovariesof the female bypostnatal day(PND)10301In males of the Sherma n stra in,plasmasampled from rats bornon day 2101gestation show a'Tsurge between birth and 2-h

ex utero ,

(2820 pglmI) falling to pre-birthlevels by4-hex utero(885 pglmI)32.A simila rsurgein plasmaTisreportedinWistarmalepups 301.50$3with pre-birthlevels of serumT tripling atbirth and then decliningsharply withinthe first 6hours.Levels returnto pre-surgevaluesby12-32.50.153to 24h301 post partum.ThesarT1Jlinglime relativetobirthappears tobecritica las the peakis notobserved in blood fromSprague- Dawteymales andfemalessampled between1700- 1800 hon the day ofbirth withoutconsid eratio nto the time of birth~I.However.serumTfromSprague-Dawteymaleandfema lepup s sacrificed at 0.5·2 hpostpartumis3limes greaterthanfrom pups sacrificed

(21)

within2-24 h postpat1umlUI.This suggests the occurrence of a postpartum surgein Sprague-Dawley neonates aswell.The surge is notlikelyrelated to factorssurrounding parturition as simlar results were obtainedin newborns takenbycesareanai,Theearly postnatalsurgein plasma

or

issuggested tobe functionalin themasculinizationof the hypothalamus32.Althoughadirect equivalency is notmade betweentheneona talages of monkey andrat,plasma androgenlevels are elevated at birthin male monkeys as discussed above for

the rat8,

In the ovariesofthe female rat,T is eitherextremely low153or not delec table until PND10:Mand in theadrenals,significantlevels are detected only afterPND17 inbothsexes:M.Althoughthefemale serumTissignifICantly lower than inmales from PND 1 up to PND 19.1." 8. 153,the amount at PND 1 is twice thelevelreport edfor every day uptoPND5118andforevery day examinedup toPNO 49...116

The sourceof plasmaor found inthepost partum female mightbe the maternaladrenalgland,which couldcontributeto the level observed at PNO 1.However,asreported for the male,asimilarbut sign ificantly smallerpostpartum'T surgeis alsoobserved infemale Sprague-Oawtey116and Wistar153pups.As well,thesex dinerenceinneonatal pla smaT suggeststhat thelevel foundin males cannot beaccounted for by the maternal adrenalgland butmustbedependent upon an additional source presentin males only.

Testosteroneis presentin thetestes oftheperinatal malen:M.l!l.1with the suggestion153thatthe risein plasma'T immediately after birthis due tothe lack

(22)

ofmetabolic clearancebythemother.The motherprovides such metabolic clearance when the fetus is in the womb.The newborn then quickly establishes a new equilibrium153.

Withinthe eNS.theleve{of'Tislowerthanin~asma2'3due tothelow diffusion/transport rate 'rom plasma to the cerebrospinalor interstitialfluid compartments117,In the PND '·2neonate,brain'Tis 15-30 %lowerthan plasmavalues inthemale and 25-70%lowerin females2'3.Asexpected , 12to 48 h afterbirth,PND1and PND2males show significantlygrea ter val ues co-caredto females 'or'T(>13x)andDHT(>4x) in bothplasmaand brain23.

Theamount ofTin bothplasma and brain of the neonatalmaleis100 times grea ter thanthe amountof E223,Surprisingly,in the neonatal femalerat,the amountof'Treport edintheplasma and brainis also greater than the aroountof E2byalact orof5- 1023.Brainlevels 01E2areverylow2'3.Although no sex differenceisreportedin plasma E2levels,the lowlevel of E2foundin the male brain is still greater than the reported value for the female brain23.Thus,the malebraincontains more~than the femalebrain,whereas both male and femalebrains contain more'Tthan E2•

Thecontinued pursuitof the ontogeny of steroidhormone bind ingtotheir respe ctivereceptors and theimplications for sexual differentiationof mating beha viorlead tothediscoveryof a neonatalbindingprotein for E2•The binding protein is nolonger presentIoIlO'Ningthree-four weeks oftife.

(23)

1.3a-FETOPROTEtN

Thehigher leve ls of~inthemale~redtothe female brainsupports the suggestion that E2isinvolvedinmalesexual differentiation(forreviewsee Gorski52,GoyandMcEwen

S;.

However.act ivationoftheestrogen receptor (EA)in the per inatalbrain is not dependentupon plasma levelsof E2•Thisis beca useE2issequesterecl in bothmales andfemale sbytheserum estradiol bindingprotein.a-felop rotein<forreview see Ueberburg12.Toran-A1lera nd1~.

Althoughvarious functions havebeen proposed forc-tetco rctetn including thatit 1)actsas a'reservoi r',releasingE2slowl y 10 prolong the half-life ofmaternal estroge ns anG'or2)providesintraneu ronaltransportof~" '163,theaccepted functionofc-Ietoprctemisthatitprotectsfemalesfromdefeminization (see Dehler~andreferencestherein)bylimitingactivityat the EA.

The propertiesofc-tetcprotem were definedusingvarious methods includingcharcoalassay ofinvitrocytosolbinding , densitygradient s,and DNA bindingviaDNAcellutosechromatography7.12O.I N.c-Fetop rcteinis different iated fromtheEAbyitssedimentatio n coefficientof approximate ly4.5S COfTllared to the coefficientof

as

identifyingtheEA7.120.124.Intheperinatalmouse7andrat brain120.12'.c-fetoorcteinisfoundat high concentrati onsintheextracellula r nuid andinneuro nalcytoplasm(see review by60.163).Aswell. c-tetoprcteln shows high capacity and greate raffinitythantheEAforbinding~,butdoesnotbind Tnornon-esnccencsteroids(includi ng DES)1.120.124.Thehigl"l capacityand

(24)

greaterbindingaffinityofc-fetc protein for E2 120.124preven ts this steroid from enter ingthe cell nucleus andactivatingtheEA.c-se tccrotemis present inboth sexesof the2Q-dayoktrat fetus1~andintheneonate atPNO 67.120.1~.It decreasesto hall the concentrationbyPNO 15,withve ry lowpresencebyPNO 22120andis undetectable byPNO 28-30124.Inthe mouse, c-teicorcietnis undetect able byPNO21'.

Because a-fetoprotein is diffuse incytoplasm of neonatal brainneurons 7.40.120.153andinparticularwithinthe hypothalamic,preoptic,amygdala,and midbrain areas120,itseems reasonableto assume that aromatizedE2wouldalso besequester edin the neonate.However, itis not known if specifcneurons containingc-tetoprctem withintheeNSareactually involved inneonatal detemlnizancn.On the otherhand,u-Ietoprcteincould beactually providing a steadysupplyinthese target neuronsbyslowlyreleasing earliersequesteredE2 40.113.Alternative ly perhaps,in the maleneonate,large enough quantitiesof E2 are synthesized in the cytoplasm suchtha t significa ntamountsescape sequestering.Regardless,the unboundaromatizedE2entersthenucleus where it attacnes totheER,thereby inducing a cascade ofgenomic events.

1.4AROMATJZ.tTlON

As indicatedinthe previous section,the highcapacity c-tetoprcten sequesters E2withinthecytoplasm7.120.113oftheneonatal brain,therebyseverely restricting actionat theER.However,

Ez

is believed to mediatedefeminizationof

(25)

the malebrain311.<10 .52.&2." (andothers,see below) andplay a role inmasculine sexual behavior in the adultrat through activationof theER10.28.30.1

61.The~that activatesthebrainERsintheneonate does notarriveviatheendocrine syslem, butrather,ismetabolizedwithinthebrain from'T,in aproce ss referred to as aromatization.Thus,althoughc-fetoprctejnsequeste rs E2metabolizedwithin the gonads,aromati zati on withinspecific areas ofthebrainprovidesa metabolic pathway that ensures local sources of E2•Testosterone,whichis notboundby c-tetcprotein. entersthecytoplasmof thecell andinthepresence of the aromataseenzyme is catalyzedintoE2Ct.7I.10 7.161.1&2.Thus,aromatasemustbe presentintarget areas where'r -derivedE2is believedto exertits effects (see Brown20,Goy&McEwenss,Hutchison&Steimer&2.McEwenetat!Mfor review).

Althougharomataseactivityis mediated via thearomatizi ngenzyme,such activityisenha ncedbythe presence ofthe AR with aromataseactivity increasing withincreasedlevelsof'T130.Botharomatase activityand aromatase messengermANA(mANA) is decreased in the hypothalamic-preo pticareaof castratedmale Sprague-Cawleyrats andrestoredbyconcurrent trealment with Silastic irrplants containing eitherTorOHT butnotbyE2I.Thelack ofa significanteffect ofE2treatment andtheequivalenteffectreported for bothT and DHT replacementin castratedmalesimplies aregu latory rolefor the ARin aromalase activityand aromatasemANA.The ARis further

r rronceted

by treatment with ananti-androgen131.Whereasifll)lan ting Silastic capsules containing'Tat the timeofcastrationrestoresaromataseactivity in adult male

(26)

ratsrelativeto cceurce . concomitanllreatmenlwiththeARantagonist,nutamide, decreases aromalaseactivity comparable10thatobservedin theuntreated castrate131.Furthermore,as cited in a reviewbyRoselli132,androge ndeficient TImmale ratshavesignificantlylessaromataseactivitycorrpared10normal littermates: the decrease is suggestedto be secondary 10the deficit in AR function132.Thus.itappearspertinenttoexamine available information on aromataseactivitywithinthehippocampus.

In adultmammals,aromataseactivityhas been reportedto occur mainly inthelimbic system,whichincludes thehippocampus(Callard24.25;cited in Brown20andin Hutchison& Steimer62).Although,aromatase activity is repo rted tobeeither negligible orverylow inthehippoc:at1lJUs,cortex1.128,1 29.131and midbrain12801theadult rat.itis threeto four-fO{dgreater inthehippocampus thaninthecortex ormidbrain1,128.It has recently beenreportedthat theintensity of aromatase immunoreactivestaining(-ir)increasesin thepresence ofthe aromataseinhibitor,R76713,permittingincreased sensitivity lo r detecting low levelaromataseactivityin specificbrainareas".As expected,Silasticcapsules containing R76713 ifTl)lantedin malemice 15 days beloresacrifteedidincrease thedensity of aromatase- irinvariou sregions 01theCNS.R76713 revealed aromatase-Ir01mediumintensitywithinthe hippocampu s and cortex01theadult male mouse".Perusal oftheir photomicrographssuggests aromatase-irwithin bothAmmon'shomandthedentategyrus(00)".The low butdetectable presence01aromatase withinthehippocatT1)Us01the adult ratis further

(27)

emphasizedin an experiment by Abdelgadir et al.1.Autoradiograms were exposedforan extended period(72V$.6 h).to enhance thevisibility of P450.\AOMwithinthehippocam;>uSI.In theneonatal rat.dataonaromatase activity inthe hipoocarre usis very limited.lowlevelsof aromatase activity1M and aromatase mRNAnoccur in the hippocampal pyramidal cell layer and DG granulecelllayerof the PND 5maleand female1Mandin the PND 6andPNO 15 male rat".

Thus, in Ihe hippocampus of the adultrat.aromataseactivity appearsto benegligibletolow1.128.129,131,with a paucity of informationavailablefor the nroeocarroc softheneonate.All houghtheARl.131.132maybefunctionalin increasing aromataseactivity, thereby optimizingactivityattheERin specific areasof the brain,such a function within the hippocampusof the neonatalrat is notyet tenable.Aninitial prerequisiteis thepresenceotthe AR within the hippccarrc us of theneonate at a timewhen aromatizedderivedE2isreportedto exertits effect.Thisrequirement willbeaddressedby the current experiment.

However,thepresence of the AR in the neonatal hippocampusis not sufficient.

It will alsobenecessarytoccrrca rethelevel of aromatase activityinthe presence01'Tand DHT relative10the castrate as has been determinedfor other brain areasl,131,l32.As hlppocarrealinformation has not been includedin experiments manipulatingaromatase activi ty inthe adultrat1,131,132.itispossible Ihatthehippocampalaromataselevels aretoolow to assesssuch manipulations.Availabledata forthehippocampus oftheneonate alsoindicate

(28)

lowleve lsof aromat aseactivityanditis not knownif treatmen t with the aromataseinhibitor,R767 13,wouldbesufficienttopermi1 evaluatio nofsteroid manipulations.

1.5 5a ·REDucTASE&DHT

Asindica ted,'T'is a major sourceof aromatizedE2inthe neonatal brain. An alternatemetabolicpathway,under the enzyme Sa-reductase.results in a sec ond biolog ica llyactive,butnon-aromalizableandrogen,OHT. Although both andro gens activat etheAA,they exhibitdifferentaffinities andbindingkinet icsas describedtater. In the rat.OHT,adrnn isteredalone,does nol promote either masculinizationofmalesexual behaviornordefernnizalion.Bothofthese appearto be undercontrolof'T'and E2(seeGorski52,Goy&McEwen55pg.

139,Plapinger and McEwen119,Toran-All erand1$3for review).The function of DHTis notwellunderstoodbutasreviewed byBrownXI,OHT activa tionofARs withinthe hypothalarne-pituitaryarea is reported toinhibit gonadotropi c hormonerelease.Ina reviewpaper,Plaping er119reportsthat OHTispotent in stimJlating peripheralreproductivetissueinme adultmale rat. As well,although DHTmay notbeinvo lved in defernn ization oftherat brain,thereis evidence lhatit mayinteract with E2toinvoke normal malesexual behavior (seeIl(for review).

Early worIl; from the197 05investigated the prese nce of 5<Neduetase activityin theratusingItlin-Iayerchromalography ofhomogenizedtissue from

(29)

different brain regionsfollowing 1-h35.31U l.162or 3-hIIincubationofbrain slices

with3H_T.The adult(PNO70-90)male35,38,11andfemal e38,11ratandferret162

showthe formationofDHTin many areasofthebrainand pituitary35.311,

includingthehippocarrous35.In general,thelevelofSc-redectaseobservedin

the adultis significantlylowerthanthelevel report edtor the neonatalrat36.91and ferr e tl6Z.Inthemidbrain311,hypotha latnJsandcortex3IUIofthe ratbrain.the highest levelsofSa·red ucta se acti vityoccurwithinthefirstpostnatalweekin bothmalesandfemales311.91.The adultvalue is observedbythe endof the first monthoflife91.The patternis slightlydifferentintheferret182,as developmental

levelsappeartoberegio n specific.Inthe ferret,Sa-red uctaseactivityinthe

preo pticareapeaks atthe endofthesecondweek oflife whereasin thecortex, higherleve lsareobse rved betweenPND 30 andPND 51162.

Thepresence ofSa-red ucta se activityinspecificbrainregions suggests theexpres sion oftheARinthesesameregions.Inde ed,incubationof brain cytosols with3H_DHTto measure bound levels in specific brainareas revealsthe interactionofOHT andtheAR.In the hypothalamic-preopticareaof the untreatedfemalemousebrain.speci fic binding assayedin cytosolsincubated with~-DHTjufTllS sharplybetweenbirth andPND9·12 withverylittle change throu gh theprep ubertalperiod7.3H.DHTbind inginthecortexofthe untreated femalemousebrainappe a rstobesimilartothedevelopmental profileofSa- red uctaseobservedinlheferret112.That is,k)wlevel sofDHT bind ing presen tin

(30)

cytosols during thefirst postnatalweek increaseduri ngthe secondweek and incr ease furtherduring thethir dweekoflife',Thisexpenrrent",however,does notfullyaddressthe ontogeny of the AR,as the devek>pmental profi leof nuclear ARswas not determined.Thus, itis possiblethat part at the eytoso lic increaseis due10 a decreasein nuclearAR.

1.6OEFElAlNrzA TION&f,lASCUUNfZATION

Defe miniza tionand masculinization willnextbe explored,notonlyto demonstrate thepossibl einvol vement atthe ARin such processesbutbecause.

aswillbeseen.Ihe data suggests anlraerdependercebetween the AAand the ERtormaximaleffect.Indeed,such interde pe ndence may extendbeyondsex differences and sexualbehavior.Foll owing aromattzano nof'T'toE2out of reach ofplas ma(but notcytoplasmic) c-fetco rctein,E2istree10 actatnuclearERs.It is obvious,therefore,lhathighe rlev els at'T'presentintheperinata lmale brain 23result in greaterERactivati on.Defeminization,defined as the suppressionof female-typicaltraits and behaviors suchastheestrogen- depend entsurge of luteinizing hormoneandthelordosisretle x,occurs postnatally.The postnatal occurrenceeffect ivelyprotectsthefemale

.n

uterotrom possible'organizing effe cts 'duetoexposure to

or

prod ucedby malelinermates".As stated by McEwen et

at. ..

andrevie wedbyOlsen11.,the aroma li zatio npathwayappe ars tobe critica ltodefemnizat ton.However,not alldata supportsthisgenerally accep ted belief.DohlerotO,whocitesmanyreports supportingthecontentionthat

(31)

aromatized E2is involvedin defeminization,alsoreports that neonatal exposure ofthe female rat totamoxifen induces defeminization in a ccee-oeoendeot manner.Tamoxifenis anE2antagonistthatbindsto.thereby blocking, intracellular ERs.CofTllaredto controls,theneonatally tamoxifen-treated females,primedasadults withE2and progesterone,do not ovulate and display decreasedfemale sexualreceptivity"0aswouldbeexpectedfrom agonistic activation01theERin neonates.However,because tamoxifenis

now

knownto elicitweak agonisticaswellas antagonistic estrogenic effects in peripheral tissue suchas the uterus,liver and

bone".

it ispossiblethat agonistic estrogeniceffectsalso occur centrally in tamoxifen-treatedneonates.If tarnoxifenelicitsits effect on deleminizatlonbyagonisticactionattheER. the tamoxtten-treatedfemalesshoulddisplaymasculinesexualbehavior when exposedto'T'asadults.The oppositeeffect occurs.These neonatally tarnoxifen-treated females, ifTl)lanledas adultswitha Silasticcapsulecontaining 1 mgtestosteronepropionate(TP) and tested threeweekslater, show a tarrcxlfe n-relateddose-dependent decreasein malesexualbehavior"0.The treatedlemalesshowdecreasedmountingandintromission behaviorrelative to control males"0.Thus.thepossibilitythattamoxifenwas acting as a true agonist atbrainERsisnot tenableat this time,However.interpretationof ER·related results isfurthercomplicatedby the recentidentification oftwoversionsof the ERwithintherat brain1Il,In,

(32)

Recentexperiments have exploredthepresence oftwoversionsofER mRNA147and fR protein110withinthe CNS of the adult rat. TheER-aversionis designated asthedassiealorprimary ERresponsibletor mostof~'sactions with the secondbeingthe newlydiscoveredEA-P version . Whereas a number at regions 01thebrainandspinalcord containeitherEA-a;orEA-j3mANA exclusively,other regions ,suchas theamygdala,bed nucleusotthestria terminalis and the preopticarea containboth signa lsIn.Withinthe nrooccerrous01the adult Sprague-Dawleyfemale,the signal is dispersed throughoutthe dorsaltoventralextent for bothEA-aandEA- j3mRNA butmore cells arelabeledlorEA-Pthan tor ER-amANA"".However,the intensity atthe hybridizationsignal lorbothtypesoftheERis reportedto be weak1<&1.Although thepresenceofER- j3mANAdoesnot ensurethe presence of the receptor protein ,ERimmunocytoctle rri stry(ICC) indicat es ER-jl- irwithintheCA 1,CA2 andDGsubfieldsofthe nropccerrcce.butnot in CA3*'.IntheICC study,only datafromthe CA2 cell/ayer is presented,making it difficultto assess the intensity of the signal forEA-Pin ttle CA1 cell layer10.Thus, itis possible that morecells labelwithinCA2 than withinCA1.Regardless,within CA1and CA2.

both interneurons and pyramidalcells containEA-fi--ireo.However.onlythe pyramidalneuronscontain both nuclear and non-nuclear immunoreactivitywith EA-jl-iralsopresent intheproximal fibers.Theinterneu rons show onlynuclear ER-jl- ir60.The distinctive partitioningattheEA-fldepending upon celltype

(33)

(pyramidalorinterneuron)suggests amJlti~erole forthisreceptor.Aswell,the presence of two forms of the ER within theeNSsuggests at least two distinct roles for this protein.It willbeofinterest to discoverif the antiestrogens,suchas tarrcxiten,elicitdifferenti al antagon istic oragonisticeffectsatthetwoversions oftheER and whether the defemniza tionprocessinvolv esjustone orboth ERs.

In spiteof the aromatizalion hypothesis,ttreAR mayalso beinvolvedin the determnizanon process.Olsen'1~foundthai theimplantation of Silastic capsules containing'Tlorthefirst 10daysoflifeCOf1'1)let elyinduced defemi nizationin femalestested as adults.Injection sof 100IJg01'Tfor 5days from PND 1 toPNO 5were lesseffective'".The'critical period'for defeminizalion,utilizingthelordo tic response ,was fur1herdefined byexamining neonatal lemalesand castratedmales givena single subcu taneou s(sc) injection of 500IJgofTPonone dayfromPND3 throug h9~.Whereas theearly neonatallyTPtreated rats (PND 3-7) displaydiminished lordosis,those treated at PND 8or 9arecomparable to untreated females31.Thus,it appears Ihal exposuretoTPfrom PNO 3 throughPND 7 maximi zesthe defem nizati on proc ess.Howe ver,i~antingorinjectingthesynthetic~aromatizable androgen,methyl trienolone(R 188 1),in neonatallemalesalsopartiallyinhibits lhe expressi on of female mating behaviors in the hormone-trealed adult'".

Thus,althoug h DHT treatmentdoesnot affect lo rdosis or preceptivebehavior '" .theinhibitionbyRl881suggeststhatdefemnizationmay notbeinduced exclusivel ybyaromatizationto E2•Severalpossibilitiestortheeffect ofR1881

(34)

are presented,includingthesuggestionthat defemnizationin theneonate could be partially mediatedbytheAR" •. Theineffec tive nessofOHT doesnotweaken thissuggestion forsevera l reasons.OHTis more rapidlyconvertedthanAl88 1 intoless potent androgens andOHT also showsless affinitythan the synthetic androge nlortheAAll•.As well,thereisautoradiographicdatashowingalack of 3H·OHT localizationwithinthe brainduringthe firstdays of life,....,suggesting thepossibility that a differenceexistsbetweenOHT andRl 88l binding to neonatal AAs.Thus,the slowerinactiv ationofRl 88 1 and greater affinityfor the ARmayhavedemon strated a directrol efortheARindefem nization.

Theexceptionsjust noteddonot,however,negatethemanyexperiments thatprovidecompelli ng evidence of the invo lveme ntof aromatized E2in deteminuanon ascitedin reviewssuchasOlsenI' •.Itsimp ly suggests that while activat ionoftheERmaybesufficien t.aettvityattheARalsoappears capableofinducingdefeminizat ion.Asindica tedearner.theARmayalsobe indirectlyinvolved indefeminizalio n by enhancingthedegreeofaromatizatio n that occurs inspecificareasofthe brainsuchasthepreopticareaand hypothalamus.However.althoughandrogenicstimula tion of aromalase has been demonstrated in adult male1.'28.13'andfemale12l.131rats,the effect of andro genon neonatal aromataseactivitywithintheeNSis still tobe determined.

Masculinizati on,theenhancement ofmale typicaltraits displa yedin matingbehavior,isalso dependentupontheactivi ty ofsexsteroids inthe

(35)

neonatalbrai n,butunlikedefemin ization,masculin izati onbeginsbeforebirth11., As reviewedbyOlsen11.,neonatallycastratedmale rats givenE2during develo pment and androgentreatmentas adultsshowmounting andintromssion behavior,butejaculatorybehavioris significantlyred uced.Similarresultsfor specificbehaviorsoccurin studi esthat replace neonatalE2treatmentwith either DHTor a syntheticnon-aromatizableandrogen,fluoxymesterone'1•.Thu s, neitherER norARstirnJlationalone producesthepattern of masculinebehavior observed in theintactmale.Malerats,castratedatbirth and given DHT(sc) rangingfrom O.1 mglday on PNO 1,3 and 5'68to 0.20mglday for10days beginning atPND 2eo,displayintromission behaviorccrrcaretaeto malesgiven eithe r ororDHT+E2. ..151.,.,Howe ver,theco~risonbetweenneonatal treatment with E2and DHTon mounting beha viordependsuponthe parameter examined,The percentag eof castratedrats showingat leastone mounting response does notdiffer betweenOHT-,TP-or eslradiol benzoate(EB)- treatment60.Likewise,hOrmonetreatment doesnot effectthe percentageof tests inwhich mountsoccur withnodifferencerepo rted between groups6O.'S1.

However,the number ofrrountsper minutein OHT·trea ted castratesis comparable to untreatedcastrates andsignifICantlylower thanthenurreer observedin EB-treat ed castrates151,Althoughoneexperimentciteszero intromissionsin male rats treated with OHTat0.50 mgldayforthe first five days of life'1,the data fromthat experiment are COJTlllicatedby toomanyvariablesto be seriousl yconsidered .Thatis, neonatallycastratedandOHT-treated males

(36)

wereimplantedwith ovaries from female littennates at fiveweeksof age and givenhor monaltreatment to elic itfemaletypical matingbehavior.Following lesting,the sameanimalswerethen sUbjected toT-pri mi ng to eticitmale- related sexua l behavior",

Asalreadystaled,neonatal treatment with eitherDHT or E2elicits equiv alentintromiss ion behaviorin maleswherease}aculatorybehavior is severely inhibitedeoQ(delayed157.158relative to neonatal treatme nt withT alone

!O.1S7.158or with DHT plus E2colTiJined151.1M .Thus,partialmasculinizationcanbe elicitedby activityat eitherthe AR or the ER whereas complete malesexual beha viorappears tobe dependentuponboth AR andER activity'".Aswell,as reviewedbyOlsen11.,theuseof antiestrog ensand antiandrogenssupportsthe assump tionthai bolh sex steroids are required;perinatal treatment wilheither alone fails to produce male sexbehavior intheT-treated adul tthatis equal 10 thatin theintactmale.

Activation of masculine traits, 'organized'in theintact neonateisalso dependentuponthe presence of sex steroids within the malebrain;again,it remains unclear exactlywhichofthesexsteroids areinvofved.Asindicat ed above,DHT binding totheAR stimulatesperipheral repr od ucti vetissueinthe adultrat119and inhibit sgonadotropic hormone release within the hypothalamic- pituitaryarea1013.Asreviewedby Hutchison&Steimer62,the AA isinvolv edin copulatory behavior inthemale rat as A1881,the syntheti cnon-aroma tizab le androgen,restores copulatory behavior in adult castratedmales as effecti vel yas

(37)

'T'10,

156.This suggests that activity attheAR elicitssuch behavior butthefinding that DHT,thenon-aromalizableendogenous androgen,has no effect on 'activating' copulatory behavior10,156negates this suggestion.It would appear that eithertwodifferentreceptors.one lor'T(that also bindsA1881)and onefor DHT(thatmayormaynotbind Al881)or aspecific conformationalchange in a single receptor.whichisnot elicitedbyDHT.is requiredto makethis suggestion tenable.This possibility is discussedmore fullyin thefollowi ngsection ('FunctionalDomainsand NeuronalLocation ofthe AA·)buthas notyet been experimentally investigated.Other work supportsthe contentionthatonly aromatizableandrogens stimulatesexual behavior in theadultmalerat.

Copulatorybehavioris decreasedin themaleratbyinhibi tors ofaroma taseas demon stratedbyintracerebralinfusionofATO28or Fadrozole:X).I66in the lateral ventral166orthe medialpreopticareaof either the intact30or'T-freated adult castrate28,166.The decrease in copulatory behavior by blocking aromatization suggests thatthis behavior is dependentupon the conversion of'T'into E2•

Indeed.unlike masculinization of the neonatalbrain.alow dOseofE2(bya factor of 100 COfT'1)ared to'T'-treatment) is effective in'activati ng'copulatory behaviorinthe adult male10.However.asreviewed by Hutchisonand Steimer52 anddemonstrated by Vagel! and McGinnis1M,acaveat exists in applyingthe eromatizaticn hypothes is.Inducing male sexual behavior incastrated ratsby the applicationofE2is dependentupon thepresenceof eitherOHT62orT1M.Even thedirect applicationof E2intothehypothaJarrlJs requires .atleast, syste mic

(38)

administrationofDHT10inducemalecopulalory behavior52,This was originally believed toinvolve theDHTeffect onperipheralsex organs such as the penis52, However,giventheeffectofRl 88 tindicated above,an alternatesuggestion madebySoderstenISSappears plaus ible;E2mayad,not onlyattheER butalso byinterfering withthemetabolism ofDHTintoinactive metabolites.

Itis possible that steroidbindingtoboth theER and the ARisrequiredfor maximal'activation'of adultmalesexualbehavior,Forexal'll>le,recent'NOfkby Vagelland McGinnis'66ind ica tes thatactivitylirtated 10either theER(viaE2- filledSftasnc implants)or theAR(via T-filledSilasticifT1:llantsplusbrain

infusion of the aromatas einhibito r,Fadrozole)doesnotrestoreejaculatory

behavior in malerats castratedas adults

'M.

However,simultaneous impla ntation of or-filled andE2-fill ed Silaslic capsulescombined withthe aroma taseinhibitor(bybraininfusion)orirJl)lanting T-filledcapsules without the aromatase blOCk,eeCOfT'4)lelel yreetceesmalesexual behavior.Howeve r,the functionofAAactivi ty in rna.lesexual behaviormaybeeven greater than formerlybelieved.

Inaneffort to addresstheconfl ictingdata regarding the invol vemen tof theAR and/orERin'acti vation'of masculinecopulatingbehavior,arecent experiment byVagell and McGinnis181assessedejaculatorybehavior inthe presence ofeithe r theERantagonist.RU58668, ortheARantagonist, hydroxyflutami de.RU 58668is reported tobea'pure'ERantag onistthatdoes not exhibit anyagonistic properties181,Thirteendaysafteri"1'lanl surgery,

(39)

100%ofthe gonadectomized'r · treated male rats administered the ER antagonist ejaculatedIS7.However,only 25%of 1he gonadectomized'Tvtreated males administered the AR antagonistdid so'81indicating thatactivityat theAR playsamajorroleinthe'activation'of malemating behavior.Althoughthe difficultyinejaculation experienced under hydroxyflutamide treatment maynot originate withinthe eNS,botratherbeperipherally-re latedbytheblockage of penileARswithasubsequentdecreasein penile spine s,there was alsoa reductionin mountingand intromitting behavior1&1.Itis of interest that the behavioral datais supported bycell nuclear exchange assaysof brain tissue.

TheEA antagonist reducedthelevelofnuclearEA presentinthe'T'-neated male(fromapprOXimately16 to7frroUmgDNA)'&1.Likewise.the ARantagonis t reduced the amount of AR present in nuclear exchange assays from 4510less than10fmoVmg DNA's'.Thus,the ER antagonistblockedbrainEAbinding but notmale copulatory behavior whereastheAR antagon istnotonlyblockedbrain AAbinding butalso inhibited sexualbehavior inthemale.

Activityat the AR may also mediale many other'organized'sexually dimorphic behaviors.Such an exa"l'ie is play·fighting,reported toinvolvethe

'organization'of neuro nalsubstrates.Intheneonatalrat,castration ortreatment

withtheandrogen antagonist,flutamide,results infemale typical levelsof play·

fightingsewhereas femalestreatedwithTor 5a·DHTwithinthefirsttwodays of life snow the masculine pattern11.TreatmentwithE2has noeffect97.Likewise,

(40)

Tfm mutations,who lack or haverelativelyfew ARs,show the lemale patternof play-lightinglilli,

Asindicatedfor'Of'ganization','activation'oftheARin theadultis also implicated in behaviorsindirectly linkedtocopulation.Forexample,intact, untreated malerats spend moretime witha'receptive'compa red to a non- receptivefemale inbehaviorthaiis referredtoas sociose xualrecognitionor partnerpreference141.Whilethemating advantagesareil'Jl)liCitinthe seeking and interactingwith a receptive versus a non-rec eptivefemale,theneural substrates unde rlyingsuch actionarenot necessari lythe same neural pathways

involvedin copulationbehaviOf'perse.However,datasuggeststhatthe

sociosexua lrecognitionpathwayinvolves activityat theAR. Treatment withthe antiandrogen,hydroxyflutamide,eliminates anydinerence in Iheamount ol tima malesspendwithreceptive~aredtonoo-receptivefemales whereasmales treated with the antiestroge nspendsignificantlymore timewith areceptive than withanon-receptive female117.Infact,antiestrogen-treated malesdonotdiffer frommales treated with'T aloneU17,Thus,the ARis involvedin'organizing' and

in'activating'the brainwith respectto masculinetraits and behavioreitherin

synergism withtheERoralone,WhileactivityatbrainARsisfunctional in initiatingsexual behavior,itis doubtfulwhetherthisinvolves mppccerrcetARs.

However,itisnotknownif partnerpreferenceisrelated toARactivitywithinthe hiopocerreus.

(41)

Beforefocusing specifically upon thehippoca~lformation and what is knownabouttheAR withinthis area,itmaybehelpful to examinethe structure oftheAR proteinas well as itsneuronallocationand other properties.Suchan examination willadd insight tothe present study.whichuses an antibodyto the ARvia ICC methodsto definetheontogenyofthisreceptor withinspecifICareas of the htppocarrous.

1.7FUNCTIONALOoMAIISANDNEURONALLOCAnoNOFJHE ANDROGENRECEPTOR

AsreviewedbyZhouIaII,theARis aligand-activatedtranscri ption al regulatoryprotei nCOf11)OSed01approximately 900amino acids in the ratand twman.Inboth species.overhalf of this sequence(fromone to approximately 540)beginning at the NH2-termnal,makes up thetranscriptionalactivation region.Thisregionissonamed becauseitis requiredfor full transcrip tional activitywithintheDNA bindingzone followingsteroidbinding151.165.Themiddle segment of approximately100 aminoacidsconstitutestheDNA bindingzone and.the hormone-binding domain,co~risedof the final 250 aminoacid sequence,islocated atthecarboxy.terminaI15I.Ie.laII.Peptide sequences used in raisingantibodie s are generallylocatedwithin theinitialtranscri ption al activation domain151.IaIIwhere antibodieswillnot interfere wHh steroid binding.

Inwhole cell ccrroeuuvebinding assayusing monkeykidney CO$-7&

CV1 cells.the hormone-bindingdOmainofthe ARproteindisplayshigh-affinity for thesyntheticandrogen,R188 1 and for thetwobiologically activeandrogens.

(42)

T andDHT72.1

. ,Theovari anhorrT'Ones,E2and progester one ,are relatively ineffectiveat binding tothe AR as lQO..fold~arexce ss is req uiredtolimit binding of3H- R1SS1 10theAR by60-S0%",Although the affinity01the AR for both'T'andDHTis repo rtedto be similar.the bindingkinetics differbetweenthe two endog enou s and rogens,Testoste ronebi nds and dissociatesfasterthan OHTas detennined intesti sandprostate tissue175,monkey COScellsillS,and in brai ncytosol s Irom female mice at PND 237,Poss ible functionalsignificanceof thisdifference,as sugge sted by ZhaoI. ,is thathigherlevels of'1'would be req uired to inducecomparable biolog ical effect s toOHT,Alternati vely,analtered confo rmation of the AR proteinunder these two steroid scould regulalethe transcription of diflerentgenes,

The locat ionof the AAwithin neurons dependsupon whether the receptor isboundto itsligand.Immunoreac tivestaining 01monkeykidn ey COScells12.ISI andeNSof Braz i lian opossums, castrated four days earlier113reveal sthatthe ARis predominantlycytoplasmicinthe absence 01androge n,with slaining large lyin the perinucl ear reg ionlSI,Theprese nceofA1881 or the addition of androgen2·hbefore fixation cause s strongnud ea rstaining6172.151.Findings extend tothe brai ns ofthe male lerret7'Il,SyrianhamsterInand Sprague-D awl ey rat7S.99,where AR.ICC7O.99.tnand nuclear exchange assayusing JH-A 1SS175 revealthatnuclearAR isdecreasedinlong- term castrates (>14days) and restored4.08h followingsys temic'T.DHTorA1881treatment7O.75.tn.Incontrast toandrogen treatment for 4·8 h.30 minutesoftreatmentreduces nudear AR-ir

(43)

densityinfive outof eight brain areas,relative tothe castrated male ferret;

however,the differencecollapsedacross brainareas isnot significa nt70.Indeed , thelack ofarapid increase in nuclearAR-ir density,relativeto theuntreated castrate,inanyoftheeightbrainareas examined30 minutesfollowingandrogen treatmentsuggests that theARdoes notsi~1ytransiocatefromthe cytoplasm tothenucleusinlong-termcastratesgiven short-termandroge n treatment70.

Howe ver.AAoccupationand translocatio n,measuredby the nuclear exchange technique,is reported toincreaseinthelong-termcastrated Sprague-Dawley rat 30 minutesfollowing treatment(sc)with androgen7$.Several possibilitiesexist lorthese diffe rences.Most obviously,the resu lts couldbe speciesdependent.

Alternative ly.thenuclearexchangeassay maylabelARs that are notidentified via immunocytochemistry.It is also possiblethat the AAdown-regulates or undergoesacontorrra ticoa lchangethatisnotreversedbytheshort-term presenceofandrogen.Such a conformational change may limitrecognitionof the proteinby the AAantibody . As well,in specificbrainareasofthemale rat.

androgentreatmentfor 1().14 days elicitssignificandygreater AR·irstaining ccrroerecto intact controls7'll.11.t9.1".Thisgivesadditionalsupport tothe suggestionthat,atleastinthose areas(including CA1~,androgen·induced AR-irwithinthenucleusinvolves more thantranslocatio nandmayinvolve androgen-inducedup-regulation ofsynthesis.

Therecognized androgens('T,DHT,and R1881)arenot1he only substancesto producetranslocationofthe AAfromthecytoplasmtothe

(44)

nucleus.Athigh doses,bothhyd ro xyflu tamide72and cypro teroneacetatet,72, recognized as anliandrogens12,also cause nudeartransport oflheARn.

Cyproterone ac etatedisplaysfurther agonist activityby inducing transcriptionin kidney COScells,whereas hydroxyf luta midedoesnotelici ttranscr ip tion,thus actingasatrue antagoni sl72Alongwiththeandrogens,high leve ls (1OG-fold molar excess) ofproge stero ne andEzresultinnucle artranslocati on of the AR and inducetransc riptio n72.

AsreviewedbyZho u186and discussedbySime ntal15\theERand proges terone rece ptor(PR) contras twiththeARas they arereported 10be predominantlynucle ar in thepresence orabse nce of hormo newhereasthe glucocorticoid(GR)receptor is bothnucearand cytoplasmic intheabsence of dexamethasone(see ZhouItl6,Simental151and references therein).Thus,ligand binding does not appear nece ssary for nucle ar locatio nofEA, PRorGR whereasit appears10bea prerequisitefornuclearlocationof the AR.

Althoughahighlevel (1QO.1lX)() foldexcess)ofEzmayinduce ARagonist aClivityin sometissues,100I-Ig(sc)doesnot reve rsea cast ratio n-ind uced increase inAR mANA observ edintheventr alprosta te,whereas'T effectively lowersAR mANA toin tactlevel s as measured 24 hta ter123,However ,a comparison betweenthe effect ofthe twosteroidsis difficultasthe dosageof or used was 20xgreater (2mg,im).'T-treatmentelicitsasimilar decreaseina castratio n-i nducedincre asein AR~NAin brain tissue,but

E.2

was not investigated123,Thus,altho ug h Ezcanmimic some of the effects of'TattheAA

(45)

inkidneyceueinvitro72,thereis presently noreport that such activitycan induce functionalchangesin prostate or brain.However,thelack01effectcouldbe dose-related.

Asindica ted abov e,the arrou nt01 ARpresentin thenucl eusdecreases intheabsence,and increasesin the presence01androgens.As well,withoutlhe presence ofits androgenic ligand.the ARdegrades relativelyQuicklyand must thenbereplacedbyprotein synthesis.Theup-anddown-regulationof theAR proteinhasbeen explo red by examiningAR mRNA.While thissignal maynotbe sufficient,itis necessaryfor production 01 the AR. Examination of the hormona l regulationofARmRNAinneural tissues were conduct ed by Northe mblot hybridization and ribonucle ase(RNase)protectionanalysis22_M.Results are rather complex as regulationappearsto be tissuespe cific and time related.As

well,datainvolving regu la tion ofthe AR mRNA maybe

coroucatec

by the

complexity ofthemessag eitself,reportedlypresentintwoforms22.

Short-term(4days)castrationoftheadultmalerat increases AR mANAin thehypotha lamic-preopticarea22,medial preopticarea58,bed nucleus of the stria terminalis51.andanteriorpituitarygland2'2with no effectontheamygdala 22.However,eithershort-termcastration (4 days)or treatme nt 01intact young adults withthe AR antag onis t,flutamide(15 mglday)for four daysdecreases hippoca mpalAR mANA13.Results following long-term (2months)castrationare similarlycorcrex and contradictory.Northem blotanalysiS suggeststhatlong- termcastrationoftheadultmale rat incre ases AR mANA inthe preopticarea,

(46)

medialbasalhypothalarrus,hippoca1T1Jus,andthepitui taryglandu.Howe ver, a late r paper from the same grou p ind ica tes that long-term castration of eitherthe

PN D25or the adult male rat decreases.ratherthanincreases,AR mANA inthe medial preop ticarea andbednucl eus ofthestriateminalis as measured by ...

situhybridizationtwofT'Ol"lltlslater58.

The short-term castratio n-inducedincreas ein AR mANAinthe hypo thalamic-preoptic area22andmedialpreopticarea58is reversedwithinone dayof acute(2lTg,sc.)treatmentwithDHT.How eve r,EBtreatment for one22.58 or five22days(10 IJglday)has no effect.Again,thereis at leasta lour-fold diHerencein the dosageused for thetwo steroids.

The effect ofhormone replaceme ntonARmANA is simila rin boththe long -ter m andshort-termcastrate in several brain areas.Forexa~le,asinthe short-term castrat e,thelong- term castration-induced incr easeisreve rsedinthe medialbasalhypo l hala rTlJs22and bednucleusofstria terminal is51by impl antation of aSilas tic capsulecontainingeither'T(7-weeks)22or DHT(2·

wee ks)w.However,the effectsof and rogenreplace ment on preop tic areaAR mA NA areless clear.As mentionedabove,castration hasbeenreport ed to have oppo si teeffect s onpreopticarea AAmANA intwo studies fromthe same laboratoryusing different method s tomeasur eARmRNA.These studi es also assessedthe effects of androge n replace men t. Intheinitialexperiment, Northernblotanalysisindicates that Chronictreatmen t with'Tdecreases22, wher easinsituhybridization analysis reveals that chronictreatmentwithDHT51

(47)

increases AA mANA in the preoptic area relative to the gonadectomized male.

Althoug hthelater studydoesnotaddresstheapparentdiscrepancybetween the setwofindings , both methods report a restoration of AR mRNA tointact levelsnSB.

Results are furthe rcoeoncated b'/theNorthern blot analysis revelationof twospeciesof AA mANA(a larger 11kb and a smaller 9.3 kb version)found withinall brain areas examined but not pituitary.The pituitary only contains the largerversion22.It isnotknownif!hesmaller versionis functional(i.e.,will it bindtohormoneand will ittranscribe ortranslate?).Giventhe suggestion that 'T'and DHT maymediate transcription of different genes,albeit due to a variationintheconformationof theAR under

or

and oHT1M,thepossibility exists that thetwoforms oftheAR mRNA regulatedifferentproteins22.In tum, the different proteinsmay be associated with different functions.

1.8ONTOGENY OF THE HIPPOCAMPALFORMATICW.

Because the presentstudyisinvestigatingthe ontogenyofthe ARwithi n neuronsof the tappocerreat area,it appears necessary to review the data on the time course ofthebirthofhippocarT1>81neurons.In this regard,it ispossible that theARispresen tin thehippocampusoftheneonateandthatitsdistribution differ sfrom that observed in the adult. Consequently,boththe time course ofthe appe arance ollhe AR anditsloca lization withinAnmon'shommay suggesta rote for thisproteinin differentiation,synaptogenesisand/orcell survivaL

(48)

WithinAmmon'shom,the earliest post-mitcnc cells are largeinterneurons arisingfrom errtlryonicday (E)15to E17within stratum (s.)cnens. s.rad iatum ande.lacunosum-molecu lar111,12.Pyramidal cell neuragenesis occurs slighUy later;themajorityofCA3 cells form between E17·E18andCAlcellsbetween E18·E 19.Pyramidal cell neurogenesis iscorrpletewithinfour days,byE202,3,11.

Becausethe pyramidal neurons sojournfromthe site oftheirbirthacrossthe tnopocarroatintermediate zone,located betweenthe future alveolarchannel and s.pyramidale cell layer,the morphogenicprofile ofAmmon's horn takes slightly longerIhan neurogenesisbutiscorcietecbyE222.3(definedusingthe first dayofgestation asEl).Thus,allpyramidal neuronsare bornandin place prenalally.

As reportedwith Iheinterneuronsof Ammon'shorn,neurogenesis of hilar cells ofthe DGoccursmainly betweenEl5-E17 and neurogenesisof molecular layercellsoccurbetweenEl 5-E19 with no postnatalgenesis11. Dentate granule cellsbegintooriginate onE17II.By E21·E22 ,granulecellsofthe dorsallimb begin10 displayamature profilewith theventral limbcells showingsigns of differentiationH.However,80-85%ofgranule cells form postnatally11.12.138with 40·50%formingbyPNO 5 and 5-10%stilltodifferentiate laterthanPNO18

Thecompletion of neurogene sisandthepyramida l cell layeralignment within Ammon'shombybirth12suggeststhat any organizationaleffects mediatedbysynaptogenesis could occurearlierinlhe pyraltidal celllaye r than

(49)

intheDG.HoNeve r,a problemexistsinthatsynaptogenesi soccurs aspartof the trieynapticcircuit.Thus.within!hehippoca rJ1)alarea,DGgranulecells extendtheir mossy fiberaxons towardthe CA3 pyramidalcellswhich, in turn extend theirSchaffercollateral axons onto CAl pyramida l neurons (see Figure 1.11.Itisobvious,therefore.that the pyrarndal cellsare dependent upon the postnatalarrivalofgranulecell axons.Such organizationaleHeets upon and pe rhaps evenactivationof theseCA1neurons would notbe expected to occur until lheseconnections are in place.possibly beginning within the first postnatal week.Indeed.Baye r and Altman12reportthat thepyramidal cell somatasare pushed furtherapartduringtheearly postnatalperiod. possiblydue to dendritic growth and thattheexpansionof the cell layer volume continues into adult life12.

Itappearsrelevant therefore,to begin explorat ionof the ontogenyoftheAR withinthe hippocampusofthe neonatalmaleandfemalefromtheearliest postnatalperiodpossible.This isa periodwhensynaptogenesis of hipoocarmel neurons and differentiation ofgranulecells canbe expected to be at a very high level andwhentheactivatingligands oftheARare known tobe present as discussed earlier.

Havingexamnedtheavailable information ontheARandits message withrespecttothebrainingeneral,the introductionwillnow focusonrelevant dataconcerning the ARwithin the hippccarrpalformation.

(50)

Figure1.1 Drawing of thedorsal hippocampus of the ratshowingboth Ammon'shorn and the dentate gyrus (OG).Ammon'shom consists ofacontinuous celllayer whichcan bedividedinto the three separate regionsofCA1.CA2and CA3.The DG consistsofa dorsal and a ventra llimb.Themossy fiber axons oftheDG granule cells synapseonto the CA3pyramidalcells,which intum,project Iheir axons(SchaffercoIlaterals) ontothe CA1 neurons.

(51)
(52)

1.9 THE ANDROGENRECEPTOR:WtrH lN THEHIPPOCAMPUS

Asindicatedin a previous section.themessage 'or ARispresentin the hippocampusof theadu ltratZV 1 152and snort-term(4 days )castration decreases73whereaslong-termcastration increases22the message.Thelevel ofARmANAisalso decrea sedintheintact rattreatedwith theantiand rogen.

flutamide.for four days(15 mglday)73suggestingthat agonisticactivi tyat the ARisfunctionalin maintainingAR mRNA levels.Alongwith the message13.the AR proteinisalsopresent within CA101theadult male ratasmeasuredby ilTlrT'lUnor eactivity21.135and autorad iograp hy13 7,Orchidect omy ofPND 26and PNO60male ratsthree daysbefore injection(iv)013H· OHT(10-20IJglkg )137or 3H_T(4IJglkg)13111-h priortosacrifice revea lslabel edneur on sinCA1andCA2 ottbetupoocarrous13&.137,Fewer cells arelabe led 'orARinCA3andCA4 with nolabelloundin theOG135.137.'ntaction 01PND26maleswith unlabeledE2,at either20or200IJglkg0'body weight,five minutesprior to 'H-OHT hasno effect on thenuclearconcentration0' rad ioactivity inCA1137,This ind icatesthatE2 doesnotinteractwiththeARinthis area.Giventhat castration of the adultmale ratdecreases hippocafr4JaIAR-irexamined fourweeks later99.itispossiblethat areducednumberofneuronsare actuall ylabeledbyautoradiographythree daysfollowingorchidect omyinboththePNO 26 andthe PND 60 male135.137.

Whilethe abovestudies onlyinvestigatedthemale.the ARisnot restricted10the mpoocarrccs 0'themalerat.Intheintact.untreatedsix-month oldrat,AR-iris presentin the hippocampus of both sexes29.How ever,sell.

(53)

differences were notinvestigatedZlI.Sex dinerenceswere investigatedwith negative resultsin a studyofhippocampalcytosolic AAs from adult Long-Evans maleand female ratsgonadectomized2-3weeksbefore sacrificeandincubated with 'H-A188195.However.itrrustbe realizedthat gonadectomy could obliterate anysex difference present intheintactanimal.Thus.sex dinerences in hippocarroatAA,evenintheadult, are snlt tc be determined.

A uniquemethod employed to investigatethepresence01theAA and the EAwithinthenippocarrp us bears exalTination.By using 3H·T inthe intactPNO 2Sprague-Oawley femaleI'"andthePNO24 Holtzmanfemale(ovariectomized andadrenalectomized atPNO23)I~rat,the 3H_T<Imarker isretainedwhether the steroid ismetabolizedornot.The'H·TIImarker.on the cmer hand,islost onlyunder aromatization to unlabeledE2,therebylabelingeither the

unmetabolizedororpresumablymetabolizedOHT,The differenceobserved

betweenthetwolabels identifiesthepresence 01theEA"'.Nohippoca~al radioactivityoccurredin the PNO2femareexaminedtwo hours following injection(sc) of either 3H_'T<Ior

'H-

'T'II indicating neither AAs norEAs were measurable at thistime

I'".

Ninety ITinutes following injection of

'H·'r

<Iinthe

PND 24 female. labeli ng waslocalized in many brain areasincludingthe hippocampuswhereas the3H

.'r

tllabel wasnot foundwithin the hippocampus l~.The lackoflabel under3H_'T'IIsuggeststhat the AAis notpresentinthe tappocarrous

ot

thePNO 24femalerat1<13,Alternative ly,perhaps atlJlisage

(54)

aromatization inthehippoca~us,al least inthefemale,is suffici entto metabolizeallthe injected

'H-T

~leading to theinaccurate assumptionof a lack of ARs.Insupportof thearomatizatronproposition.autoradiography of thePND 26 male Holtzmanrat.castratedthree days beforereceiving anivinjection of3H_

DHT and sacrificedonehourlater,showsa highlevel of labelinginthe tuppccarro cs137.The radioacti velabelis COf'rl)arable to thatinthePND60 male '37.It is nolcurrently knownif results relateto ahippoca~1sex difference,10 diffe rencesinthe sensitivityof thetwomethods or 10other variables suchas age.

TheARappe ars tobepresentinthehippocalll)USofthe fetal.neonatal andadult roonkey.Howe ver,a cautionarynoteis thatsuchinforma tion generally comes from studiesinvolvingoneor two animal s ateachage examined.Inthe adult malecynoroolgu s monkey(Macaca fasdcuJariS),hippocampalneuronsare net consistently labe{edbyAR-ir21.Ukewise,althoughthe AR-irlabel is reported tobewidely distr ibutedthroughOut Ammon'shorn andtheOGinthe rhesu smonkey(Macacamufatta).it isalsowidely disperse dwithineacharea27.

Thiscontrasl s 10 findingsin the adult ratwheretheARis localizedin virtually everypyramidalneuron

at

CA 1 andCA2 as identifiedbyirmlJnocy1OChemistry

21.1:15and autorad iographyusing3H-DHT137.As well,corrcared toreport s of the

presenceofthe ARin theDGof the adultmonkey".itis reported tobeabsent intheOGof theadult rat135..137.

(55)

The AR is also reportedly presentwithin the diencephalon of the letat and neonatalmonkey27.31.57.101.However.neonataldata is severely limited and an ontogenicprofileisnotyet available.Thefull gestationalperiodforthe monkey isapproximately166dayslqj.Thispermitsa CCIfIllatisonofdata between the monkeyandthe rat. Examinationof various areas of the diencephalonofthe rhesusmonkey between gestation day 50to150 reveals both AR-irvand the presenceof ARwithin extracted cytosots(using~R 1 881)57.In the cynomolgus monkey,theARis presentin nuclearpellets (using~.T)101extractedfrom the hippocampusof theintact male and femaleat day 122 of gestation101.No sex differenceinhippocafT\:lal ARoccurs inthe fetalrhesus57andcynomolgus101

monkey.Intheneonatalgonadectomized maleandfemalecynomolgus monkeys,the hippocampal ARis present at PND 6·8as labeled'T and DHT are extractedfromnuclear pellets60 minfollowinginjection15.As well, the

'ejccort ical'hippocafTlluS from one0-3month-old female rhesusmonkey

exhibitshigh affinity and capacity for androgenbind ingrelativetoother safT1lled brainareas31.Theaffinityandcapacity wasdetermined by saturationbinding analysisconductedoncytosdfractionsusing3H-R18813'.

Thus,althoughtheAR appears tobepresent inthe hippocafTlluS ofthe adultrat andmonkey,the distributionand quantityappear to differ.As well,in the hippocampusoftheneonatalraland primate.thereislimitedinformation aboutthe presenceoftheARwithnodataavailable ontheontogenyof this receptor withinthisbrainarea.

(56)

Althoughsex difference sin hippoca fTl)al AR havenot yet beenreported, activation of ARsdependsuponthe presence of its activatingligand,eitherTor OHT.Brain levels of both of these ligandsarehigher in the male,suggesting thatARactivatio n would alsobegreaterinthe male.Thus,any function mediatedbyhippocampal ARs couldpotentiallyelicit oneor moresex differences.II the refore appears relevan ttoexploreinforma tion about sex difference s with respecttothehippoca rJ1)Us andmpoccarrcer-reretedfunction s.

1.10SEXDIFFERENCESANDTHEHIPPOCAMPUS

Sexdifference sin spatialbehaviorintheMeadow vole(Microtus pennsyfvanicus).,."',ratla l21,monkey'andhuman1111and theccrtetancne withthesize otthe hippocafTl)uS65.6&and the DGgranule celllayer121suggest that'Tmaybefunctional in behavior thatis onlyindireclly related to copulation.

Forexample,orappearstoberelated to sexdifferencesinlearni ng reported for the monkey',rat53.6U&.lll.Ial21and human".

Sex differences inthedevelopmentoflearning abilities repo rted inthe rhesus monkeyare postulatedtoberelatedto the presence ofgonadal steroids (lo r review seeI).The argument is made thatthepresenceofandrogen promotes maturationof the orbital prefrontal cortex,as males perform an'object discriminationreversal'task,showntobe dependent uponthis brainarea, signit icanll ybette r thanfemales.Thissex difference occurs onlyintherelatively young monkey(2-3 monthold) and disappears with maturationofthefemale

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