Androgen Producing Cells in Polycystic Ovaries

Margaret M. Convery

Department of Anatomy

McGilI University

June 1989

A thesis submitted to the Faculty of Graduate Stuùies anù Research in partial fulfillment of the requiremcnts

For the degree of Mal.ter of SCIence

(~, Margaret M. Convcly

ABSTRACT

A single injection of estradiol valcrate (EV) produces a cascadl' ni spl.'nlic

hypothalamic and pituitary impairments, culminating in polycystir ovalll'S (peO)

(macrocystlc PCO). Chmnic exposure to physiologle,11 Ieveb ni 17-B-eslnllhul (1; ,) ViiI.

subcutaneous implants (Silastic R capsules) also resuJts in :t pulYl"ystic ovanall

condition, about which less IS known. The plasma gonadotmplIl patterns aI l' Vl.'ly

different in these Iwo models, as IS the hbtoloh'Y 01 the p()lycy~tlc ()varie~. Sinn' III

both varieties ot PCO tbecal relis and theral dellvat ives arl' vely promllll'Ilt, tlw<;l'

cells have been studied at the light and electron mllTOSCOple kw\. Flve IIldll"eS 01

cytodifferentiation and stL'foid synthetlc activlty have hcen CXiII11I1ll'd, mdudlllg,:

alkaline phosphatase expression, LH hindlllg capacity, mitochondnal arl'a, "PIt! dmplet

area, and cell size.

Thecal cells from healthy and atretic tollicles III hoth macfIlcyst Il" and

microcystic ovaries show intense alkaline phosphatase actlvJty, nllllleroll~ 1 J I-hlllding

sites, and the ultrastrllctllral characteristlcs of cell~ actIve III stef(ml(.:~ene~ls.

Macrocystlc and microcy~tlc thecal cells exprcs~ Ilttle alkalme ph()sphata~e il ·tlvlty,

possess a negliglble numher of LB himlmg ~Ite~, amI contillll thc largc~t amoullt (lI

Iipid with respect to hoth the size ot the dropkt~ and ahundancc. Thl'sl' ilJ(.IICl'~

suggest that cystic thecal cells are ~terOJd()genrcally inactIve. ('y~tlc !olllcle" appear to

be the result of an atretic process. In hoth the macr()(.:y"tlc and IllICf(Ky"tl(' I11mleJ,

the proce~s of atresia ~eems to involve a rrogre"~lve deactlvatlon 01 thccal ('l'II", wlth

cystic follides representing the completlon of the rroce~". The large ~r:te (lI

macrocystic folhcle~ may prevent them t'rom collap!-.mg, and thll~ they rcmalll rn the

ovary. Mlcrocystic folhcles, on the other hand, !->ecm to he folllde~ III a tellllllléll ~tatt'

of atresia, prior to collapse.

Secondary rnter~titial cell c\u~ter:-. in hoth mélcrocy~tlc and mlcrocy"tlc ()vafll:~

show intense alkallt1e phosphata!-.e activlty and the large~t numher of 1 J f-hl/ldlllg ~Ite~.

This suggests that these celb are the m()~t ~cn"'ltive tn trorlc horm()ne ~tlmuJatl()n, and

the most actively androgenic. The u1tra..,tructural charélcten\tlc~ of the~e celh, le.

mitochondrial and lipid droplet arca, al'io !-.ugge~t thls. Â!-. the alkalme ph(J~rhata~e

activity élnd nurnher of LH hinding sites between secondary interstitial cell c1usters are

more varia hIe in EV-treated ovunes than in the E2-implanted model, and as E2-

implanted ovaries contain a more extensive population of secondary interstitial ceI1s,

the androgen levels in the /llIcrocystic condition would be expected to be higher than

in the l11acrocy~tic condition Differences hetween the EV-trcated and E2-implanted

polycystic ovaries may retlect ditferences in the pattern of tropic hormone stimulation.

Narnc: Margaret M. Convery

Title of Thcsis: Androgen Producing Cells in Polycystic Ovaries

Dcpartrncnt: Anatomy

Degree: M:t~ter of Science

Résumé

Une seule injection d'oestradiol valerate (EV) proÙll\t Cht'L Il- rat unl' St'lll' dt'

détériorations hypothalamiques et hypophy~alrl'~ spéClliqul's qUI CUhlllnl'llt dall~ hl

formation d'ovaires polycystlque~ (PCO) (peO l11acrocy~tlqUl'). Unl' t.'\pll~ltHlIl

chromque à des niveaux phy~lologique~ de 17-B-oe~tradl{l1 (1: .. ) via dl'~ Iml'Iilnl~ MHI"­

cutanés (capsules de POIYÙlIlléthylsIluxane) a aU~~1 pour ré~uItat 1" t0I111alHlIl d'llV:url'~

polycystiques, condition ~ur laquelle 011 a 1l101ll~ d'lIllormatlol1 Il y a tllll' gWlH.k

différence entre les proflb du taux pla~ll1al((.llIe de~ g()nad()lrllphlllt'~ ainSI qllt'

l'histologie des ovaires polycy~tlqlles de cc~ deux ,lldt'les, ('Ol1lllle k~ l'clluk ..

thécales et leurs dénvées sont très proémlllcntes pour chaqul' type dL' PP), lT~

cellules ont été étudiées en IllIcroscopie optIque et élect r<llllqUl'. Clllq IIHlln'~ dl'

cytodlfférentiatlon ct d'activIté synthétique des ~ténlllk~ ont l'Il' l'Xilnllnl'~, IIlClu.1Il1·

l'expressIon de la phosphatase alcaline, la capacltl" de h:lI~on dl' 1 J l, la "'UpCI "t'Il' dl'"

mitochondries, la superficIe des gouttelette~ lipolùes ct la dlllll'Il"'J()Jl de ... cellule,>

Les cellules thécales provenant de lolllcule ... ~:lIn~ et atr{-~ltlue .. d'ovau n

macrocystIque!:> et Illicrocy!:>tlqucs pré~entent une <Ictlvlté inttll,>e de 1 .. phmphaté\~(,

alcalll1e, de nombreux ~ite~ de liaiSon de LI l, et le!:> caractémtlqllC'''' ultl:l~trUl'tuIale~

de cellules active~ dans la production de ~ténmle~. Le,> ('elluk~ thrcales

macrocy!:>tlques et micr()cy~tiques manJfe~tent une hllhle actlvltt' dl' 1;1 plm"phata .. l'

alcalIne, pmsèdent un nOll1brL' négligeable de .,Ite,> de hal,>on de 1 J 1 cl ("()ntlt'nlll~lIt

la plus grande quantIté de lIpide en ce qUI concerne la dlmen'iIOI1 ct l'ahondillll'l' dc~

gouttelettes. Ce~ imhces ~uggèrent que ces cellllk~ théc;:lc~ Cy~tlqlll':-' sont Inactive ...

en ce qll1 a trait ù la ~ynthè~e des ~térO!des, Le,> folllcule<, cy<,tlqlle., :-'l'lllhlent ('tre Il'

résultat d'un pr()ce~~us d'atré'ile. Dan., k cas du mod0k macn Il) ,>tlque comme d,Ill"

celui du modèle mlcrc)Cy~tJ<lue, le proce.,.,u,> d'atré'iie dont k~ folilcuk ... atré:-'I(IlH.~.,

représentent l'achèvement, ~emble ll11pliquer un raJcntl'i,>cment progre,>,>1f de~ cellule<.,

thécales. La grande dlmen:-'Ion de~ folhcuk~ lllacrocy~tlqlle<, pourféllt pr6vcrllr Icur

désagrégatIon et, par con~équent, ils restent dan~ l'ov,lIrc. Par contre, k~ f(llllclde,>

microcystiqlle~ semblent être à un ~tage tcrmlllal d'atré~lc avant km d6~agrégatJ(H1.

t ....

Le), groupes ),econdaires de cellules mter),titlelles des ovaries macrocystiques et

mlc[ocy),tiqlH.!), pr6),entcnt une activité inten~e de la phosphatase alcahnc et le plus

grand nOl1lhre de ~Ite~ de h(lI~on de LH CeCI ),uggère que ces cellules sont les plus

),cn"ihle), a une ~tlJll'J!(ltlon hormonale trophIque et les plus active~ dans la formatIOn

d'amJf()gene~, De m6me le~ caractén~tlque~ ultrastructurales de ces cellules, ie, la

~lIrerflCle de), Il1lt()chondne~ et de!'> gouttelettes hpo'ldes, suggèrent ceci. Comme

l'activIté de la ph()sphata~e alcalIne et le nomhre de )'Ites de lImson de LH entre les

grollpe~ ~l·c()ndéllre ... de cellule!'> inter!'>titlelles ),ont plu), vanahles dans le~ ovalre~ traités

:'t l'I:'V que déll1~ le modèle Implanté avec l'EL' et comme les ovaIre), IInplantés avec

l'L2 cOlltlennent ulle populatIon plu~ étendue de cellules ~ec(lndalres interstitielles, on

s'attend :'t ce que Ie~ nIveaux d'androgènes de la condition microcy~tique SOient plus

ékvl'~ que CCLIX de la condItion macrocystique. Les ditférences entre les ovaires

tl;llt~~ il l'EV ct l'eux IIllplant6 avec J'E2 pourratent refléter des différences dans le

profil de ... tllllulatIO(1 de ... hormone~ trophIques.

Nom: Margaret M. COIlVCry

Titre de la thèse: Les Cellules QUI Produisent L'androgène Dans Les Ovaires P()lycy~tJq lies

Drpartrment: Anatomie

Dipltmlc: Maîtn!>e es Sciences

ACKNOWLEDGEMENTS

1 wish to thank the following persons:

- Dr. James Brawer, for hls !.upervision and gllldance, 1 1 cndll'ss paliencl' and

encouragement, and for tll1ancial support.

- Dalia PIcCInni-Chen, for ha advlce and lechnical as!.istancc.

- Dr. Mike LallI, Jeannie Mui, Pat Hales, Mlchal:1 Kraetz, ilnd ail 01 the EM

Luh technicians, for teaching me electron n1J(:ro~coplc techniqucs élnd lm tbclr

patience in helping me solve techl1lcal probkl1l~.

- Dr. Riaz Farookhl, for prepanng the radlnligand.

- Mr. A. Graham, for his as~i~tancc with photography.

- Len Wedlich, fur hi~ assistance 111 prepaflng the photogmplllc ligures.

- Michele Piotte, for tramlatlllg the ah~tracl for IbiS thcsl!..

- The Center for the Slmly of Reproduction for their fïl1é\llcial support.

- Gramlma, for her emotion,lI aml financIaI ~uprort.

- Dad, MOITI, Mauleen, Mlc!1;ICI, Kathleen, John, Shell:l ilfld Bflilll, lm the

countless phone call~ and Ictter!., and for thcir conlllknce and ~lIPP()rt.

1 also wi~h to thank Jaillit Gill for hi~ encouragement, hl~ help, and for lm.

friendship which has made the ddflcult days more bcarable.

TABLE OF CONTENTS

INTRODUCï'lON ...................................... .

The Hypothalamic-llypophYl>eal-Ovarian Axis ........................... 4 Role of the Hypothalamus ln Femalc Reproductive Cyclicity ........... , 4 Role 01 the Antcrior Pituitary in Fcmale Reproductive CycIicity ........ , 4 Role of the Ovary ln Femalc Reproductive Cyclici tY ................. 6

Exp~flmental Manipulations Leading 10 PCO ........................... 11

EV-Induccd peo Moùd . ....................................... 13 llypolhalanlll: anù Pituitary Dcfccts A<;sociated with EY-Induced

PCO ... . ...................................... , 13 Dcvdopmcnt of a Hil.tologically Definitive, EY·Induced Polycystic

Ovary .............................. ............. 13 Jli~tol()gical Fcaturcs of an EV-Imluced Poly<--ystic Ovary ............. , 15 EndOl.:nne rcature:. A"socialed with the EY·lnduccd Polycystic Ovarian

. . .. ................................. 17

('11I01l1C E:.tradlol (LJ Exposure-Induced peo Modcl ..................... 19 lIypoth,damlc and PltUitary Dcfccts A<;sol-iated with ~·Induced Polycystic

Ovanan Comhllon ................................... , 19 IIJ~tologlcaI FeatUJc~ or an E2-Induccd PolycYl.tic Ovary .............. , 20 Emlm:nnc Fcaturc:-, A~~m:iated with the E2·Induced Polycystic Ovarian

C'ondltlon . .. . _ .. ....................... . • • • • • 21

Slgndïcancc of lhing Two Modcls To Study Polycystic Ovarian Conditions ...... , 22

Thc~ i~ PI ()Po~," ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... , 24 Ralion,t1e .. ............................................ 24 Pr()pn~al. .............................................. 27

I\IATFRIAI.S AND METIIODS .... . .. ................................. 30

Animal 11()t1~ing and Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 30

Eslradlol V.t1er.llc-Trcatetl AnimaIs .................................. 30

Ânllnall> RClI:ivmg E~tradiol-eontaining Implants . . . . . . . . . . . . . . . . . . . . . . . .. 31

Experil11ent.II Proccdure - AIJ-.aItnc Phosphatasc Study .......... . . . . . . . . . .. 31

Experimental Procedure - hCG St udy . . . . . . . . . . .. . ........ . Preparation ami Administration of hCG Radioligand ...... . Preparation of Tissues for Radioautography ........ . .. . Electron Microscopie Radioautngraphy ............. , .. Electron Microseopy ..... . ..... . ........... . Determination of CcII SilC .................... . . Analysis of Data .. ............ ............ ..

RESULTS .......................................... '"

Histologieal Features or Polycystie Ovaries '" .......... . . Light Microscopie Description of Macrocystic Ovaric~ Light Microscopie Descnption of Microcy~tic Ovaries ..... .

Alkaline Phosphatase . . . .. . ........ " ............. . Alkalinc Phmphatase: Primordial and Pnmary F(lllidc~ .. , . Alkalinc Phosphatase: Sceondary and Graallan r(llhdc~ .... Alkalinc Phosphatase: Atrctic Folhdes. ..... . . .. . Alkalinc Pho~phatase: Corpora Lutea . . . .. .... . .. Alkalinc Phosphata~c: Seeondary Inter~tltial Cclls " . . .. . Alkalinc Phosphata~e: Prc~y~tic Follicb . . .. ... . .. Alkalinc Phosphatase. Cy~tie Follidc~ . . . . . . . . . . .. .

.

· · · .

~ ... ~ ... ~6

~h

H ~H

N

41

4 ... 44 45 4h 4h 46 47 4X

Electron Microscoplc Cytology or Thecal and SecondaJY Intcr~tltial Cdb. 4()

E-;tradiol Valcrate Trcatcd Animais - Macrocystlc Ovarian Condition 49 Light Microscopic Analy~i~ of CcII Sile ...... 49 Electron Microscopie Identitïeatlon 01 ThecaJ Cell~ 49 Electron Miero~copie De~criptlOn 01 Thccal/Secondary

Inlerst!lial CcII MorphoJogy . .. ...... . 51 Electron Microscopie Analy~i~ 01 Llrid Droplct Area . 51 Arca of Mitochondrial prorile~ . . .. . 54 Analysis of wI-hCG Bmding Slte~ lhing l'. M. Ralhoautography 55

~ Implanted Animab - Micro(,y~t!C Ovarian Condltl()n . . . . . 5()

Light Microscopie Analy~l~ of Cell SII:e . . . .. . 5()

Electron Mic/O.\copi<.: IdentificatIOn oJ Thccal ('dh . )() Electron Microscopie De~crI ption 01 Thecal/Secondtlry

Interstitial CcII MorpholoI,'Y . . . . . . .. . 57 Electron MiLloscopic Analy~i~ of LI»/ù DlOplct Area . 5X Arca of Mitochonùrial proriles ... ' . , . . . . .. . . 5X Analysb or 12"I-hCG Bmding S/tc~ U~Jng E M. Radlo(tutography 5C

)

l\facrocystic vs. Mlcrocy~liC Thccal and Sccondary Jnrer~ti(ial CdJ!> ,.. 6J Analysis of CcII SiLe ....... , ......... "., . 62 Lipid DropJct Arca ..... _ . . . . . . . .. .... . . . 62 Arca of Mitochondrial Profiles ............ , . . . 62

DISCUSSION .....................................................• 63

Alkahnc.: PhC)~rhatase Exprc.:~~ion ... . . . . . . . . . . . . . . . . . . . . . . . . . • . . . . . .. 63

CcII Sile am! UI-flmùing Capacity .................................. 65

Mllochondriai and Llpld Droplct Profile Arcas ..................•....... 68

Ilow arc.: c.:y~t~ relatcd to other follicular structures? " . . . . . . . . . . . . . . . . . . . .. 71

R l'HJ~EN(,ES . . .. .... . . . . .. .................................... 74

1·1 ( ; 1 J R r :S ........................................ 87

INTRODUCTION

Cyclic reproductive function in mammals dcpcnds UpOIl mtl'grated interactions

between the hypothalamus, the pltuitary and the ovaries. IkclIlISl' the mcnstrlléllcyclc

(humans) and the estrous cycle (rodents) are truc cycll's, a first cause, il'. a cause

without an antecedent cause, and a final end Il'sult cannot hl' H.Il'ntllll.'d. Cyl'hclIy is

dependent on the balanced rcciprocal interaction hdwl.'l'n thc hralll, the IHtmt'lI)' :Iml

the avaries. Alteration or lo~s of endm:nnc 'Ignab at any kvL'l along the

hypothalamo-hypopLy~cal-g()nadal axis Ieads to il dl~rllptHH1 of the balancc of thl'

system, and a cessation of cycltcity a nd ovula t ion. J\ l'Oll1\1H Hl con,cq ucncc uf Sllch

disruptions i~ Polycystlc Ovarian Di~case.

The polycystic ovarian condition (PCO) is a state of anllV1Ilatoly ilcycheity

occurring in a \VIde range of I1W111111:11lHn specle~. Tilts reproductIve é1IHlIlWly

encornpasses a range of expre~SI()n!-., élnd is no! a slllg/c dl,c;l~e entl!y. l'he '>1:lle 01

chronic anovulation I~ respomlble for the infertillly élS~()Cléltl'd wI11l PCO. P( 'OIS :1

major cause of infertJlJly in WOl11cn, cattle, pork ami !-.hecp. It 1<; chal actclll'ed hy Ihe

presence, within the oVllfie~, of llluitiple, large cy~tlc lollrck..,.

The anovulatory state ni the ()varte~ in hlllll:ln\ wlth PC<) IS pre~lIl1lahly :l

response to the altercd gonéldotrorll1 Signais bCll1g relea~ed lrom the pllll/lary, (Yell

et al., 1970). In our laboratory, wc have generated chronic p()lycySllc ()vallan

conditions in the rat by administcring a single large dmc ()f e~tréldl()1 v;J!crak (LV)

(Brawer et al., 1978; lIemJlling~ ct al., 1983; Schlll~ter ct aL, 1 <JX4), or hy c11101l1C

exposllre to physiologicélileveis of ] 7-f3-cstradiol from subcutaneously implanted silastic

c<lpsllics (Brawer et al., ) 983; WIlkinson et al., 1983 and 1985). In the EV-treated

m()(lcl, the EV injection engcnders an intractahle hypothalamic lesion (Brawer et aL,

) <J7H, /(mO éII](! 1983), which I~ correlatcd wIth, but not proven ta be causally related

I(), ail ()h~clvcd delccllve hypolhalamic regu!éltion ot Illteinizing hormone releasmg

hormone (UIRII) (Simard et al., !9K7; lllrnere et aL, 1988). As weil, impaired

pltllilary lull'lIIillllg hormone (U I) ~Iorélge and release (Simard et al., ]987; Carriere

d aL, JC)XX) (l['ClIr. Thc cascadc of hypothalamic and pltlllttlry defects which develop

to!!mvlIIg 1 :V-illjl'ctloll IIltlllwtely kild 10 the e~tahlishll1ent of a unique pattern of

gonad{)tropin ~eLrctll)JJ tn which the ovary ultiméltely responds hy developing a

polycystic IlH\lphology (Gros~cr ct aL, 1~187; McCarthy et aL, 1987; McCarthy and

Brawcl, 1 ()X<)). In this model, naltrcxone administration or hemi-ovariectomy indu ce

.lItClilliolls 111 the PCO-as~oclated LH pattern (Farookhi et al.. 1985; Carriere et al.,

l')XI)) <lm! n':~1111 III !'.igllli iea nt 1 eslora t Ion of ovarian Il I~ tology and functlon, suggesting

1 Il li t 1 hl' p()lycySI le oV;lfIa Il morphology 1 cpresents t he re~ponse of a normal ovary to

;1 SI1L'C1lle, ; Il)J1 ()I 111 a 1 hm 111011:11 situatlOlI.

(,hrunie exposllle to phy~iol()gical levels of 17-,8-e~tradiol via. a silastic implant

katls 10 the dc\'elopmcnt ot a hypotlllllall1ie le~ion and alteratiom. in hypothalamic

opialt' hinding (Wllklllson et ;11., IWB and 1985) which resemble those induced hy'.::.\-'

iI1Jl·etioll. ;\~ wl'II, a '.Inique paltel n of gOlladotropm relea~e and polycystic ovaries are

l'st:lhllshed wlthlll eight wccks lollowing implant placement (Brawer et al., 1983;

2

McCarthy and Brawer, 1989), The plasma gonadotropin patterns il ml the m'i11 ian

histology, however, differ considerahly trom those Sl'cn 111 thl' EY-illlllll'l'd polyryslÎl'

ovarian condition (McCarthy and Brawer, 19~9). Smcc removal of Ihe slltlslic Il11planl

containing 17-,B-estradiol is followed by restoration of normal ovarian lllOl phology ilml

function (unplIblished results), the polycystlc response 01 the ovaril's to chro\1le

estradiol exposure also like Iy represents the rcsponsl' ni iln L'ssent la lIy nOl ma 1 OVi\I y

to a specifie, ahnormal pattern ot hormonal stimulation, as is Ihe case in the HY­

treated mode!.

The focus of this thesis is on the ovary of rats wlth polycystlc OVi1i1i1l1

conditions. An analysis ot the polyeystic ovaries resulting lrom holh estlillllol vall'Iille

and 17-,B-estradiol t reatment will he m'lde. Speellica Ily 1 he Iole olt hl' a mit ()gell

producing ce Ils, néll11ely theenl celb ilnd !'.ccol1dary intcr:-.liti.J1 t'l'lb, in t he:-.e n '1H.IIIIIln:-.

will be exammed. Ot partlcular il1terest arc the hypel t\llpllled :-'l'COl1llilly II1tl'rsllllal

cel1s and folllLular theeal cells 1)L~I()nglng tn :-.tructurcs 1I1l1quc to a polycystic ()vilry,

incIlIding preeystic and cy!'.tic follicles.

As an lInderstancling of the phenomenol1 or cycIJcity 1<, cr ilieal tn asse:-,sIllg and

understanding any dl:-.ease of reproductive mfcrtIlity, lIlc1udlllg peo, il hllL~1 di:-'Cll!'.:-'HIII

of the mie ot the hypothalamus, the (Il1lerI()r pitultélry glillld, (1 III 1 ()f Ihe OVilrIC!'> ill

cyelic reproùuctive fllnctiol1 wIll hl' pre:-.ellted. Thereétlter, the ,l11ll11al Il1Cllleb ll!'>ed t()

stlldy peo and the expenmental proposab ot thi!'. the:-'I:-' ,Iwll be di:-,cll!'>:-.ed dlrectly.

3

The Hypothalamic-Hypophyseal-Ovarian Axis

Role of the Hypothalamus in Female Reproductive Cyclicity

A collection of neurons in the medial basal hypothal '11US (in humans) or in

the medial preoptic area (in rodents) synthesize a decapeptide known as luteinizing

hormonc relca~ing hormone (LHRH), also known as gonadotropin hormone releasing

hormonc (GnRH) (sec Knohil, 1980, for review). This peptide hormone is released

hy thc!-.c nellrolls mtn the pitllitary portal circulation (Dierschke et aL, 1970) in a non-

contlllllollS, pllisatile milIter (sec KnobJl, 1980, for review). The LHRH is

slIhscquenlly tr;ln<.,ported ln the anleflOf pitllltary where il induces the synthesis and

rdea~e 01 two c1mely rclilled glycoprotein hormones: Illteinizing hormone (LH) and

tolltele stlll1l1l:ttmg hormone (FSH) (Matllso et al., 1971). The signaIs which initiate

the clrchoral pulsatile discharges of LHRH onginate within the central nervous system

(Carmel et al., 1 (76). However, these hypothalamic signaIs are modifled by exposure

to cstradiol ;lI1d olher ~tcroidal factors synthesized by the ovary (Fink, 1979).

Circulating stl'Ioid horlllones of ovarian origin feeLl hack on the hypothalamus

plOvidlllg hoth positive and negative influences on the l'requency and amplitude of

1 J IRII rckase dllflng the course of the cycle (Butcher et aL, 1974; McEwen, 1979;

Rill1lCy ct al., 1<)~7).

Roll' ot the Ântenm Pituitarv in Fel11ale Reproductive Cyclicity

'l'hl' gOI1i1dotlOpes are rcgulated hy complex signaIs l'rom bath the hypothalamus

lInd tllL' ovary. The re~J'0nse of the gonadotropes to the hypothalamic signal, ie.

4

LHRH, depends upon the nature of the pulse parameters l'Omprising the signal. il'.

amplitude and frequency. With respect to the amplitude. as the dOSl' of 1.lIRII

released increases, the quantlty of LH and FSII rekased hy gonadot mpes also

increases in a c1assic dose-response fashioll (Knohll. 19l{O). 'l'hl' system IS saturahk

in that there is a maximal LHRl-I dose ahove wllll:h no addrt ronal LI 1 01 FSII

secretion will he elicited (Knohil, 19l{O). The tempolitl pattern of the hypothalalllil"

LHRH signal can cause suppre~s\On 01 hoth LlI and FSII secretion. stllllulall' FSII

secretion while suppres~il1g LlI secretlOI1, or stlllllllate the rdl'a:-.e III hulh

gonadotropins (ie. LH and FSJ 1) (~ec Knohil. ICJKO tOI rl'vlcw). Whlk l"'''pl'r IlIll'nlal

results suggest that LH secretion IS entirely depcndent on the cndogcl!ous LlIRII

pattern, there seems to he a second factor IIcting ~ylll"rglsllc;tlly wlth 1 J IRII tn

regt/Jate FSH secretion (Culler and Negro-Vifl:lr, 1 <)l{7) ;I~ 1 hel e <Ire JIl~laJlce<; III whlch

FSH secretion is dissociélted from that of LI 1 (cg. the 1()lhcul;1/ pha~c 01 the llIel1~truill

cycle or the diestrous phase nt the estrous cycle). 'l'hi.., tact()r has Ilot Yl't heell

identified.

In addition to modulating the hypolha\;llllic LI IRI 1 pattern, ovanélll :-tcl(lIlb

modify gonadotropin secretion hy exertJJ1g hoth p()~itlve ill1d l1l'gallve Icedhilck ellecl:­

directly at the level ot the gOlladotrope. The pmitive kedhilck cflccb (lf ()Vilfl:l1J

estradio1 inc1ude sensltlzation 01 the g()lladotrope~ 10 LI HU f, ~() thal lower

concentrations of LHRH are needed tD elJcit éI re~pon~e (Ramey et al., 1 9X7). 'l'hl'"

positive or "priming" etfect contnhutc ... 10 the LI f ~lIrge wlllt:h ()cc.:ur~ JlI~t prim 10

5

ovulation (Butchcr et aL, ] 974). The negative feedback effects of estradiol on the

pltuitary occllrring dunng the dlcStrollS phase of the estrous cycle (GaIlo and Bona-

Gallo, 1<)~5) arc related 10 inhihition of gonadotropm secretion (Butcher et al., 1974).

Rolc of the Ovary in Female Reproductive Cycliclty

The role of the ovary in the reproductive cycle of mammals is twofold,

involvlIlg: J) thc prmlllction of mature eggs, and 2) the elaboration of steroid and

peptide hormoncs whlch II1flucncc ~exual bchavillur and which con tribu te ta the

rcgulalHlIl 01 hoth hypothalamic ,ml! pilllitary fllnction.

The procc~s 01 lolhclilm dcvelopment, ovulation and the development of

corpora lut ca 111 the mamm,lIian ovary IS weil known. The ovary contains a pool of

rcstll1g ~erm ccll~, known as oocytcs, containcd wlthm follicles. These resting oocytes

arc SllJ 1 Olllldcd hy li single layer of granuln~a cells. a ba!\ement membrane, and

st rolll<l 1 ccll~. Together. thcse cellular a!\~,()ciations constitute a prImordial follicle. Of

the many tolhclcs cOlltalllcd within the ovary of an adult cyc\ing mammal, only a

Il'strictcd Il li 111 IX' r oVlllatc in rcsponse to each gonadotropin surge. Those follicles

whlch do not devclop to thl!'> ovulatory state Will undergo a degenerative process

klloWII as a t rcsia.

GIO\vth and J11atUl1ltloll of tllllicles is dependent on gonadotropin stimulation

(Rirhards and Mldglcy, 1976). The dynamics of folliclllar growth and cellular

dilTerelltiatlllll, will ch arc nitlcal to the continued developlllent of a follJcle, depend

llpOIl spt'cific chal1gt'~ 111 tlle content of receptors on folliclllar cells for both LH and

6

FSH. Furthermore, these changes In receptor content are tlWlllsel\'l'S rq~lIlilll'd hy

specifie interactions with estradlol and FSH (Richards and MH.lgky. 1976). \1111 IlItlon

of follicular growth, ie. the transItion lrom thc prImordial to tht' pllmary stage. orrlll~

spontaneously (Pederson, 1(70). Thcrcattcr. howl'\'l'I. rontllllll'd g\(nlh and

maturation of follicles is dependent on gonadotropins (Richards allll Mldgky. 197h).

The transition from the prImon.lIa1 to the pnl11ary ~Iagc IIlvolvl's plOllkrallon

of the granulosa œil layer and the orgal1lzatlol1 01 tlll' Mlllolllllling sl\O\l1alrl'lh JlIIIl

a thecal layer external to the hasement membrane (Pl'Il'r~. 1 ()()lJ). 'l'hl'

cytodifferent iation of 1 he stroma 1 cc II~ mlo t hera 1 ce 1 b IIIVO Ivl'~ the ilcq 1I1~1 1 iOIl (lI

steroid hlosynthetlc enzymes and LI 1 reœptOls (Enck,>oll ct al., )lJX5), and Ihl'

development of a specialized ultrastructure COI1Slstlllg 01 exlemlVC ~11l0()lh l'l1d()pla~nlll"

reticulum, ahundant Illitochondria \VIth tllhlliaf CII,>t:ll'. ami 11l1111l'I()U~ II/ml dropkb

which contain the precursor (Il'. chole~tl'Jol) lor ilndrogcn hlmylllhl"~J:-' (F<lwl"l·tt ct :11.,

1969; Encbon et al., ] <)f{5). The néltllre 01 thl' :-.tlllllllu~ 1I111lallllg the dllkll'l1lli1tl<lJl

process is unknown, huI IS believed to be dirccted hy ~Ignilb elllan:llllIg fI()1l1 thl'

developing follicle it~elf and tn he Illdcpendcnt 01 pillllt:l/y ~Igllal\ (1 ~ncb()11 cl al.,

1985). LH stimulation ot tully dillerentlated tllecal ccII'> rl"~I1II~ III ccII lIypertlOphy

and anclrogen productIon (Rlcc and Savard, ]W,(); Ryan :lIld l'cIro, l')()(); and F()rtlllle

and Armstrong, 1(77). l'Ile granulma cell~ of prImary lolllcle,> conlilln l'SII rect'pt()J\

(Midgley, 1973; Nimrod et al., ] <)74; Zcleznrk ct aL, ] cJ74). l'SII ~tllllulatl()11 01 Ille~l"

cells results in proliferation of the œlb lorllllflg the I11clllhrana grilfllll()!'>;1 (1 ~.,hk()1 ;lIId

7

Luncnfcld, 1(72), incrcased arornatasc activlty and, consequently, increased estrogen

production (Ullenbrock and Richards, 1979). The estrogen produced by these cells

is not synthe~lzcd de novo. Rather, granulosa cells convert C-19 androgens of thecal

origm tn CI H c~trogcn~ hy dccarhoxylation and aromatization of the "A" ring of the

sleroid moleclI le.

As folhculogenesis proceed~, the cells of the mcmbrana granulosa continue to

proldenlle III rcspon!'.e 10 FSI l ~tJll1l1latlnn, addltional thecal stern relis prohferate

and dllfcrentlatc (Encbol1 et aL, IlJH5), and !'.terOld blo~ynthe!'.is mcreases. Eventually,

the fnlhcular 1ltlld !'.ecrcled ÙlIflllg the growth process cOéllesce~, and a tluid-filled

anlral spacc i~ formel! (McKay et al., 19(1). The lolhc1e IS now called a secondary

or antral folhck. At thl~ ~tage oi lollicular gmwth, the perimural granulosa cel1s of

:s kw lolllcll's WIll :sequin: LI 1 rcceptor!o. as a result ot the !o.ynergistic actions of FSH

:1I111 l'!'.tradlol (Rieh:1J d~ et aL, 1 <)7 ô ). Most follicles, however, do not. Thase

sl'cond:lry follie/e!'. wlth ahlllllbnt LH receptors on both granlilosa élnd thecal cells are

thl' Olll'!'. wlllch rl'spol1d to thL' LI 1 ~urge by ovulatmg. ul1lkrgomg Illtelllization and

lOI Illlllg C()I pora lutl'a. whlle lolliLIc~ whose granlllma cells have gamed kw or no LH

rl'ccpIOl!'. rl'~p()lld hy lI11dergolllg alresla (Midgley et aL, 1974; Richards and Midgley,

197(l ).

Folhcles I1wy hecome alretle at any stage ot their development (Uilenbroek,

\Volilnsl'n and van der Sehout, 19~()). Initiation ot the atretic process may be due

to L'UI1l'r :In llladcqu:Ite llumher ot gonadotropin receptors, or to the fallicle being

out of synchrony with gonadotropin stimulation (Richards amI M idg\ey. 197h). l'hl'

pattern of atresia is not umtorm and de pends on the stage 01 difkrenltatlon ",hll'h

the follic1e ha/> reached at the tlllle when Il begllls tu degclll'lale (By-;ko\', )t>79). In

general, the process ot follicular atresla involves the death ot the lHWylt' and gl.llllllm.lI

cells. Interestmgly, the cells uf the theca lIltelna survIve the dl'gcllCltlllVC pllll"e~'l'~

aftlicting the rest ot the folltcular relis amI llndcrgo a 111:11 ked hypl'I tlophy. Thc~l'

hypertrophied cells then settle in the reglol1 ot the olll lollICk and gl\'l' II~l' 10 li

population ot cells called ~econdmy II1tcr~tltJ;t1 l'clis (KlIlg~hllly. \t)J!). na\V~tlll ,1Ill!

McCahe, 1951; Renneb, 1951; Guraya and Greellwald. It}(ll'-:; :ml! Ikalll'~ly. 1972),

Cytologically and blot:hcI111cally, ~ee()l1dary IIltcr~tltl:tI n:lI" l"Ol1tllllll' to nlllhlt

propertles of their prccursor, thL: thecal ccII, Hl IIJ:lI thcy 111<11111;11/1 thl' ~pl'crall/t'd

ultrastructural chélructcristlc~ of :Ictlve ~ter()Jd()gcl1lc ccII:-. (("IIltIH'I!'. ;JIIl! (Jn.'l'I1. It)72;

Mori and Mélt~lIlll()to, 1973; AJ-Mchdl, J<>79; alld E1ICk'()Il. 1 <)l'-:.1), they n,l'Il"" 1 Il

receptnrs, and they contlllue tn rc~p()l1d tn Lli hy produClllg élIllItO\tl'lIl't!Jtlllt' (RICt'

and Savard, 1966; MeN atty d al., J <n9; and Fnck"'()Il, 1 <)X3),

Regardle~~ ot the c.kvt:l0plllcntal ~tatc 01 OV(lrJ,lIl 1()lhcul"r ~trllcturc~ or tlH.'1/

derivatives, the elahoratlOl1 of stcroid IH)rl11(}Ilt:~ hy tht: folllclIlar ccll~ dl/ring folheulal

growth, by cells ot the corpu~ lutculll !ollowlIlg :In ()Vl/I:IIIOIl t:vcnt (Ilotahly C\trogcll

and progesterone), and hy g()llad()troplll-,cn~lllve cdl ... of the Illtcr~tltllllll will III tU/II

affect the nature 01 the hypothalalllle :Iml pltultary :-.ign:t1 to whlch tlley rc"'polld hy

the teedback mechanbm:-. de:-.cnhcd.

<J

Inùccù, the phcnomcnon of reproùuctive cyclicity is complex, anù depends

upon intncate reciprocal interactIons between the hypothalamus, the pituitaries and

the ovaries. How can this balance whlch support cyclicity, once established, be

ùi!'.rupteù to cffect the development of polycystic ovaries'!

10

1 Experimental M,I- :, Itions I.cllding to PCO

A wide range of experimental manipulations rl'~lIlt 111 pl'r~l~tl'l1t vaginal

cornification, anovulation and polycy~tlc ovanes bllatl'fally 111 lahoratory rats. For

eXélmple, treating neonates wlth testp~terolle «(,or~kI, Il)(ll)). adults with

dihydroeplllndro~terone (DIIEA) (Parker and Mahe~h. 197h). or hypothYlOld rats with

human chonolllc gonadotroplll (hCG) (Copmallll and Adal1l~. 19XI). ail rl'~lIlt 111 a

polycyst1c ovanan condition. A~ weil, cOllditl()n~ 01 plOlongl'd hght l'Xpll~lIll'

(Camphell and Schwartz, 19XO; and Daal:e and Parlow, 1 (ni). lutcll1l/.1ng hormonc

relea~ing hormone antibmly adl1lllm.trèltinl1 (Pupklll ct al.. p)~n). 1I11plilntatuln ul

estradiol henzoate <.:ry~tals lOto the (lnterlor hypothillillllll: ,lIea (Kaw,lkilI111 a III 1

Visessuvan, 1(79), antenm hypothalanllc dcalkrcllt.ltloll (Ililla~/, 19()(»), ilgll\g

(Ascheim, 1(76). aÙmll1l~tratl()n ni e~tradlol valerate (Br:lwt:r et :11., 1<J7X ami 1 <JX(l;

Mohhs et aL, 1 <JX-l), and !-.lIbclItaneoll!-.ly Illlplantcd C~tl allJ()1 (1 ~J-C<lntallllllg chrolllc

release l'ap~lIles (Brawer ct aL. IWB) will also result 111 l'CO.

In ~pite ot thc hypothaléll11lc and pltllltary Il11p:llrmellt~ prodllccd hy the~c

different treatments. many of the~e rats wlth peo arc !'.tlll capahle 01 1Ilfl1111lg cllfpora

lutea in re~pnn!'.e tn certain cxpcnmental m<llllplll:ltUJIl!'.. The p()lycy~tlC ()vane~ III

hoth the neollatally androgcl1lzed and the L-:V-I/lduccd nH)deb lorm corpora lulea III

response tn an LHRH challengc (Hahn and McGulre, 1 <J7X; /lemmlllg ... et aL, Il)X1).

The induction of corpora lutea I~ élccompallled hy the dl ... ;lppearancc ()I cy~tlC 1()lhclc~

from hoth ovane~, prohahly hy ra~~ive coll1pre~~I()n (('ol1very ct aL, 1 <JXI).

11

1 {ypothalamlc ami pituitary pathologies, however, persist. Hemi-ovariectomy results

in restoratlon of normal ovarian histology and ovulatory cyclicity within one week in

the EV-treated rat (Farookhi ct aL, 1985; Convery et al., 1989). Again, hypothalamic,

pituitary and hormonal ahheratlons persist. In the DHEA-treated, constant light­

induced and ETlmplanted models, normal ovarian morphology and ovulatory capacity

are re~tored tollowmg the cc~sation of treatment (Parker and Mahesh, 1976; Daane

and Parlow, 1971; and unpuhlished results, respectively). The fact that cyclic

reproductive tllllction can he re~tored tn a polycystlc ovary. despite contÎnued

dyslunctlon ot the hralll and pltllitary, further support the notion that the hlstologically

ddlllitive pnlycystlc COl1lhtllln (PCO) d()c~ not retlect an mtnnslc ovarÎan pathology.

ln our laburatory, two modeb of the polycy),tlC ovanan condition have heen

extensivcly studtcd and charactcnzed: EV-mduccd peo and peo which results from

chrollic exposure tl> physlological leve\s of 17-#-estradiol. These two modeb Will be

lIsed in this thc~ls. The specitic change~ m the hypothalamus, pttuitary and avaries

in these Illodels are descnhed helow.

12

EV-Induced PCC Model

Hypathalamic and Pituitary Defects Associated wlth EV-Imlul'l'd peo

A single, 2mg injectIOn of a long actmg estrogcn, estradIot valcrate (1 ~V), glWIl

ta young cycling rats will mdure a ~erics ot progrc~slvc changl.'s "Iollg Ihl'

hypathalamo-hypophyseal-gonadal axis Ieading to anovulalory acychl'lly, pl'lSl~ll'lll

vaginal carnification and the devetopment of polycystlc ovallCS Wllhlll tWCIlIY-l'lghl

days (Brawer et aL, ] 9R9), In the hypothalamu~. 1l111ItI!ocal IcSIOIl~ 111 the all'lIall'

nucleus (Brawer et aL, 197R and 19RO), an tncrea~cd llulllhcr 01 naloxo\1l' hll1dlng

sites (Wilkinson et al., 1 Wn), and detectlve rcgllla tl(lIl o! 1.11 R Il 'l'ri l't Il)1I (SIIU,lI li

et aL, 19R7; Carriere et ai., IW';R) have hcen ob~crvcd Pltllltary abhl'latl()i1' III ri Il dt'

a decreased pitllltary LH content (Schllbter ct al., 19R4), dl'Cll'a~l.'d L1I1U 1-,lll11l1laled

LH secretion (Hemmmgs et aL, 19X3), and a depn.:~~ed Humher (JI L1IRII rl'l'l'plll/~

on pituitary gonadotropes (Carriere et al., 19XX). Ba~al l'OllcclltratIOI1~ ()! 'l'lUlll 1.11

at eight weeks is low, whIle that ot ~erllm FSII l~ In the hlgh-l1orm,d lange (1 kllll1lll1g.,

et aL, ]984; Schlllster et aL, 19R4; Simard et aL, I<JR7), a:-. I~ the pltllllilry l'Sil colltent

(Simard et aL, 1987). Thus, ail tacet~ ot LH prOllllctl()n ilppear 10 he !-.ekrtlve!y

comprornised, whi1e FSH production IS mtact.

Development of a HIsto]oglcally Detll1ltlve, EV -1 nduced p()lycy~IIC ()vilr)'

The sequence o! event~ Icadlllg to the dL:ve!opment and L:xprL:"~((lI1 o! polycy"llc

ovaries seems to occur in two pha~e~ (Brawer et aL, 19k(», The !Jr~t, cIlLolllpa:-,.,lllg

the penod of time t'rom the Jay of EV-injectIon to tour weeh thercaltcr, I~

13

characterized by a steady decrease in the population size of follicular structures of ail

ranges of diameter, accompanied by a decrease ln ovarian weight. Although follicular

loss occurs unrformly ln follicle!> of ail diameters, a selective enhancement in atresia

amongst large !>econdary tollicles is observed dunng this period ln time. By as early

a~ 16 days pOlIt EV-Injection, an absence of Graafian follic1es is observed. By 28 days,

corpora lutea are ah!>cnt, and large, tluid-hlled cy!>tic t'ollides are established bilateral1y

(Brawcr ct al., 1 97X, 19H6, 1 lJHH élnd ] 9H9). Another mteresting morphological

ch:tractcn~tlc ot thc~e ()V(lne~ I~ thc pre~ence of lutelnrzed granulosa or thecal cells,

occurnng III c1l1~tcr~ clrcul11~cnhed hy a ba~ement membrane or diffusely scattered

throughout the ~troma. Although the~e are most likely secondary mterstitial cell

c1l1ster~, they are ohservcd much more frequently m these ovaries versus a normally

cydlllg ovary. Grm~ly, the ovane!. are smaller than their age-matched, untreated

cychng countel part~, and can be turther dl!>tmgui!>hed nn gross examination by the

prcsence ot very prol11l1lcnt, large, tlUld-tilled protrusions,

Thc second ph;tsC 111 the development of the polycystic ovarian condition,

la:-.tll1g tWill day twenty-clght to day tJfty-~lx, is characterized by the achievement of

a ~teady-!>tate populatHlIl ~Ii'e ut ail folhcle~ élnd by non-selectIve atresia, The degree

01 i1trl'~l<l III the allècted tolhcles dunng thls phase, however, is suhstantially more far­

readllng.

As the rhanges Jcadlllg to the elltahlishment of cyStIC follicles in the polycystic

l'omlJtio\1 of an e~trmhol-valerate treated ovary are stahilized by fiftY-SIX days following

14

hormonal treatment (Brawer et aL, 1986 and 1989), eight week nvaries will he USl'd

in this study.

Histological Features of an EV -ln_duced Polycystlc Ovary

There are three types of follicular structures which art' unique ln this IW­

induced polycystlc ovary. The most characteristic of thcse, the lollicular cyst, l'onslsts

of a highly attenuated granulosa cell layer, a hypertrophied thecal l'l'II layer, and

occasional macrophages lining the antral surface ot the Jl1emhrana grilnulosa. The

second type, the precystic follicle (Brawer et al., 19R6 and 19X9), Il'semhles the cyst

except for the presence of irregular and often rather extcnsive patches 01 drgellcraring

granulosa eells along the antral surface of the mcmbrarw gWllulosa. In IIghr

microscopie sections of epon-embedded ovaries, the thecal ccII laYCI of thcsc Iwo Iypes

of follicles appears to consist primarily ot large, polygonal œlb III1l'd Wlt Il IJpld ghll'>t~,

with a few small fusiform cells scattered In betwcCI1. The tlmt! type lIf follwlIlm

structure found in these polycystlc ovanes, identllïed hy Brawer ct al. ( 1 ()X<J) as a Type

III large follicular structure, is characterized hy a large antr,,1 ~pace lïrClll11~crihed by

multiple layers of healthy granulosa cells. The thecal ccII layer coll~l~b of il dIffuse

layer of small, fusiform cells, resemhling those charactcflstlC 01 healthy, devc\oping

follic1es. The Type III large follicular structure dittcrs lrom Graallal1 1()llic\es 01

normal cyc1ing ovaries in that it is a larger structure, mltotlc 1 Igurc~ ilr~ ~cell il !1l()l1gst

perimural granulosa cells, and they do not contain ova (Braw~r ct al., 1 ()X(); COl1vcry

et al., 1989). Paradoxically, the Type III large foilicular ~tructure IS th~ only f(Jlhcular

15

structure unique tn the polycystic ovary in which granulosa cells bind LH (hCG)

(Brawer et aL, ] 989) and is, therefore, the only structure which cou Id be readily

luteinized followmg an LH surge. However, the Jack of corpora lutea in these

polycystic ovanes suggests that this does not ordinarily happen. The nature, function

and mie of tl1ls folllcular structure remains to be elucidated.

Many of the cy~ts observed ln eight week ovaries exhibit patch es of

degcncrating granulosa cells at intervals a)ong an atherwise healthy-looking mura)

IllCmbl ana granulosa. This fcature is not characteristlc of cysts from later time

mtervals (HeI11I1lIng~ et al., ] 9R3; Schulster et al., 1984; Brawer et al., 1986). The

role atresia plays in the two phases of development of polycystic avaries, and the

appearancc ot these cysts, has raised speculation that true follicular cysts may be

prodllcts of partial or arrcsted atresia (Brawer ct al., 1986). This possibility is further

SlippOi tcd by the ob~ervatlon that the hyperthecosis characteristic of cystic follic1es is

:Ilso charactenstic of atretic ~cc()ndary follicles (Byskov, 1978). Furthermore, the

prescnce of severcly atretle secondary follic1es at fi ft y-six days, similar ta those

ohserved at em 11er time mtervals following EV injection but rarely if ever seen in

oldcr cystic ovaries, sllggests that atreslél plays an important role in the ultimate

mmJclling of tlllS polycystlc ovary and that cystic follicles may be the consequence of

an arIest of the atretIc process (Brawer et al., 1986).

16

Endocrine Features Associated with the EV-Induccd Polycystk Ovarian Condition

The histological evolution of polycystie ovarics in this mmlclcoinndcs with tlll'

development of a unique low amplitude, high frequeney pattern of LIt. Although the

pattern is pulsatile, the regularity in the frequency and the ill11plitudl' of the pulses IS

markedly different versus those charactellstic of pulses ot thc estlOus phase of the lat

estrous cycle (Grosser et al., 1987). The FSH pattcrn is i1lso pulsatllc ami is l'IllSl'ly

synchronized with the LH pattern (Grosser et aL, 19H7). l'hl' charactellstll' pattclns

emerge between two and four weeks after EV trcatmcnt, ie. cOlncll.lcnt wlth thc

emergence of defmitive cysts wlthm the ovaries. FlIrthermore, thcy arc ohsl'fved III

aU animais with estabhshed PCO, regardless ot the dllratioll 01 thc mlldltHIIl

(McCarthy et al., 1988). Based on these tïndings, Gros~er ct al. propose tllat thls

uniq ue plasma gonadotropin pattern, rather t1mn the mca n ~crllJn gOl1adot roplll

concentration, is causal to the estahlishment and entie:!1 tn the m:tmtcllilllCC ot the

polycystic ovarian syndrome in this mode!.

Radioautographre studies conducted on EV-treated ()vane~ t() asse~~ tllclr

sensitivity to LH mdieate that cyst formation e()ll1clde~ wlth the Im,~ ot ~en~ltlvlty (II

precystic and cystic folhcle~ to LH. Bmding ot /25 I-hCG (ail LI 1 an:t1CJglll: 11\t..:d III

radioautographic studies hecause ot it~ ~tabihty [Richard~ élnd Mldgley, j(J74» III

precystic follicles IS seant and IJmlted tn the cclb 01 the thecéI IIltema (Brawer ct al.,

1989; Richard, 1988). Radlolahelled hCG htndmg in cy~tlc t()lJicle~ i~ ab~e/lt ilJllong:-.t

cells of the membrana granulosa and is rare or altogether ahsent ln the tileca interna.

17

In contrast tn precystic and cystic follic1es, cells of bath the perimural membrana

granulosa and ce Ils of the theca interna in type III large follicular structures show

intense and dense patches of labelling. These structures, then, are presumably LH­

sensitive. LaheJJing of clusters of secondary interstitial cells is highly variable in these

polycystic ovaries, varying t'rom dense ta absent. The theca interna of normal and

atretic follicles hind hCG. The degree ta which they bind hCG is similar ta binding

reportcd for ana)ogous structures in a non-cystic ovary (Bortolussi et al., 1979; Shaha

and Grccnwalù, 19R2). Thus, the precystic and cystic t'ollicles are truly unique, not

only structurally hut, as weIl, in their LH binding characteristics. As weil, despite the

-;tructural hOlllology hetween cystic theca and the thecal ceIls of atretic follic1es, there

must he tunctional differences hetween thern as they demonstrate marked differences

in thcir célpacity to hinù hCG (or LH) (Richard, 1988; Brawer et al., 1989).

18

Chronic Estradiol (E21 Exposure-Induced PCO Model

Hypothalamic and Pltuitary Defects Assnciated wlth E,,-Indul'cd PoIY('ystlc Ovarian Condition

The extent to which chronic exposure to physiologieal plasm:t COllù'ntl:llions

of estradioJ, delivered by means of chronic release implants, altcrt thl' hypo1ilalill1111-

hypophyseal-ovarian axis of rats to yield a polycystic ovanan colllhtHH! IS kss llclilll'd

than it is for the EV-treated peo model. Within tluce wccks followl\1g placl'Illl'nt

of the E.,-containing implants, cycliclty IS arrestcd, and pcrslstent vaginal rornltlratHlll

and anovulation ensuc. A dctinitivc polycystlc ovarian COl1lhtlon I~ Iully c~tah"shcd

by at least eight weeks after placement ot the silastic implant. ThIS l'Olllhtion pClsists

unless the capsule is suhsequently rl'moved (unpuhllshcd result~). Intact or

ovariectomized E 2-implanted animaIs dl'velop a lesion \11 the anterlm hypothalamw.

and express enhanced hinding of opiate~ in the antenor hypothalamus wlthm ten

weeks folJowing initléltion of treatment (Wilkinson et aL, 1 <JX3 and 1 9X5), a~ do 1 ~ V-

treated rats with peo (Brawer et aL, 1975). The~e estradiol-Illduccd e\kct~ cali he

expressed in castrated rats, wherl'as the development 01 the hypothalalllic Jl:ttllOl()gle~

following EV-mjection requires the presence 01 the ()vafle~ (Brawer d al., 1 <)XO).

The pituitary defects associakd with thi~ mode! have Il()t yct heen HJclltllled,

although a decrease m pituitary welght IS rcpnrtcd hy W"kin~()n et al. (1 <JX5) tell

weeks after initiation of chronic estradiol exposure.

19

HistoJogical Features of an ETIndueed Polycystic Ovary

The ovaries trom E2-treated ammals, in contrast ta those from EV-treated

ammals, are large and smooth surtaced. Non-atretic large secondary follicles are

rarely scen in these ovanes. Corpora lutea are altogether absent. The eystie follicIes

ohserved in these ovanes are markedly different than those observed in the ovaries

01 EV-treated animaIs. They are more numerous, smaller, and are charaeterized at

the light microscopie Icvel by an unusually thick theea interna comprised of

hypertrophied, polygonal cells filled with lipid ghostll. The thecal cell layer in these

cysts is approximately four to five tlmes the thiekness of that in the eysts of EV­

treated animais. The granulosa cell layer eonsists elther of a ~ingle layer of cells or

several layers ot degeneratmg cells. Beeause of the dlfferences in the size of the cystic

structurel> ohserved 111 the ovanes ot these two different peo models, the eysts from

an EV-induced polycystlc condition may he suhsequently rcferred to as macroeysts and

those from a polycystic ovary induced hy chronic exposure to 17-t1-estradiol as

microcysts.

As was ohserved in the EV-induced polycystie ovaries, numerous c1usters of

sccondary Il1terstitial cells occur scattered throughout the stroma. However, these œIl

c1usters me larger and more frequently ohserved as compared to an EV-treated ovary.

Althollgh the ~ize and shape of some of these clusters suggests that they may be

dcrivcd flO111 collapscd cysts, the large size and frothy appearance of the ce Ils of sorne

of thest' ccII c111~ters as observed in epon-embedded light microscopie sections, due ta

20

their engorgement with lipid ghosts, suggests Cl different origill (McCarthy and Brawcr,

1989; and personal observations).

Endocrine Features Associated with the E2-Induced Polycystic Ovarian Condition

Unlike the EV-treated rat with peo, the E2-lfl1planted rat exhlhit~ a cnmplc'\

plasma LH pélttern. McCarthy et al. (19R9) demonstrated two typcs 01 epismlir Lli

secretion in these animaIs. The first COIlSIStS of short, frequcnt, sharply dclmcated

low amplitude pulses. This pattern is very similar to fhat sccn conslstently III rats

with EV-induced PCO (Grosser et aL, 19R7). Thesc short, low amplItude pulsl's arc

interrupted at regular intervals by the second type ot pulse, con~i~tl\1g 01 epl!.odcs 01

LH release of high amplItude and long dunttlol1. Although FSII patterns gl'IllTally

conform with the LH patterns, definitive FSH peaks are not oh!.ervcd to corrclate Wlt h

those of LH (McCarthy et al., 1989). ThIS IS in contras! with the ~yl1chJ()n()w.

gonadotropin patterns reported for the EV-mduced mode! 01 l'CO (Ciro!.!.cr ct aL,

1989) and is suggestive nt a second mechant!.m involved 111 FSII regulatlol1 111 !hcsc

E,-implanted animaIs.

21

Significance of Using Two Models To Study Polycystic Ovarian Conditions

The charactenstics ot the EV-induced and E2-implanted models of polycystic

ovarian condition are ~imllar to two expressions of cystic ovarian disease in humans.

The mnrpholo!:,'Y ot the EV -induced polycystic ovary resembles what is called a

multilolhcular ovary, seen ln women wlth hypothalamic amenorrhea associateu with

welght loss (Adams ct al., 19R5). The ovaries of women wlth thls pathology contain

several very large cystic folhcles, and do not exhlhlt stromal hypertrophy. In contrast,

the hUlllan p()lycy~tlc ovary ln the cléls~ical Stein-Leventhal syndrome is enlarged,

contaill~ Illultiple ~111i111 cy~t~ ilnd an exten~lvely hypertrophied stroma (Adams et a1.,

1 <JX6). Thl~ IS Sllllilili 10 the ov:\nan morpholo!:,ry in rats recelving subcutaneous

implants contallllllg estradiol (Brawer et aL, 1983; McCarthy and Brawer, 1989).

ln addition tn the ~lIl11lanties m ovanan morpholo!:,ry, serum LH patterns in

hUl11an and rat cy~tlc oVélnan conditions show similarities. In hypothalamic

amenorrhea, the Lli pattern IS greatly suppressed, exhibiting only occasional low

amplitude pulses. Snl11e patients also experience larger pulses at mght (Khoury et

aL, 19X7). The c()rre~pnndlllg polycystic condition in the EV -treated rat i~ associated

with a Ilighly suppresscd pattern ot LH release, consl~ting of low amplitude, high

t rcqul'Ilcy plllsl'~. 1 n ma rkcd contrast to the pattern of gonadotroplll release in

patlCl1t~ wlth hypothalalllic <Imenorrhea, women with Stein-Leventhal syndJ'Ome express

an cxaggcratcd pattern of LH rèlease, consistmg of high amplitude pulses arising l'rom

a higher l11t'all nadir (Rehar et al, 1976; Waldstreicher et al., 1988). Although the

22

frequency and nadir of the highly irregular pattern of LH secretion ohst'rVL'd III

animaIs with a polycystic condition induced hy chronic E 2 l'xposure <.IOl'S not

correspond to the pattern in the human, the large LH surge~ observed ail' L'onsidl'lL'd

to retlect a highly stimulatory pattern of secretion (MeC'a1 thy and BI aWL'I, 1l)~9), onl'

which may or may not he comparahle to that 01 the Stt.'in-LeVL'nthal !\yndlotnl'.

In addition to the parallels hetwecn ovarian Illorphology and plasma

gonadotropin patterns in hurnans with hypothalamic amenorrhca :lI1d rat~ wlth an I~V­

induced polycystic ovarian condition, hoth disorders ean he amchorated hy hloekadmg

the opiatergic system which is involved in regulatlol1 of LlIRII nellllll1!\ 111 Ihe

hypothalamus (Khoury et al., 19R7; Wtldt and Leyendccker, 1')X7; Cal rien= et al.,

1989).

23

1 THESIS PROPOSAL

Ration? le

Although there is gond reason ta implicate the alterations in hypothalamic

LlIRH or pituitary gonadotropin secretion as the underlying cause for the generation

of ovarian cy~t~, the role played hy the ovary in maintaming the e~tahli~hed polycystic

condition C:lnl1ot he considered msignitJcant. In the case of human peo, ovarian

wedge re~ectJol1s have hren used sliccessflllly to temporarily Hlleviate the acyclic

coJ)(.htiol1 of the~e patients (Goldzieher, }9Rl). 111 rat~ wlth éll1 EV-induced polycystic

ovarian cOl1<.htlon, oVlllatory fllllction resume~ following hemiovariectomy, despite

continueù dy~f UI1CIIOI1 at the hypothalamic and pituitary Jevels of the axis (Farookhi

et aL, 19K5; COl1very et aL, 19XY). Removal of the E2-contaming sila~t1c implants after

the polycystlc morphology has heen estahlJshed will also he followed hy a restoration

of morphology al1ù lunctlo!1 to the previously acyclic, anovlllatory ovaries (unpllhlished

results). The lact that expre:-.sions of reo are reversihle strongly suggests that:

changes in ovarian functlo!1 in reo are not the cause of the dlsruption in cycJic

reproductive IUl1ctlon; anù polycystic ovaries are not an expression of ovarian

pathology. peo may he simply a potentléll developmental condition of a normally

tUllctiolllIIg ovary whlch IS a manifestation of a primary pathology elther al the leveJ

ot the hypothalamus or pltultary. If this is in faet the case, what is a polyey~tic ovary'!

The exi~tenœ of specialized populations of ovarian interstitial cells is

phYSlologically ~igniticant hecause the androgens they secrete are of major importance.

24

Androgens are obligatory precursnrs for estrogen hiosynthcsis (Baggctt ct al., Il)5h:

Ryan and Smith, 1961: Enge1, 1973), estrogens heing essential tor !lm III a 1 Il'pIOllul'tlVl'

function. In addition, androgens tnduce folltcular atresla (Paync ct al.. t 956 and Il)5X:

Louvet et al., 1975). In pnlycystic nvarian disease, the ovam's alc anovlliatory and

acyc1ic. Furthermore, follicu lar atreSta scems to play an lin pm ta nt roll' III thl'

modelling of a pnlycystic ovary (Brawer ct al., 19R6 and 19X9). What roll' du thl'

interstitial ce Ils play in the deve10pment and maintcnancc of l'CO'!

Interstitial cell populatIons arc located tn huth the thcca Întcln:l of lolltrles and

the interstitml compartment of the ovary. Ali II1tcrstltial relis ari~e /rom li popul:llHlII

of unspecialized mesenchymal l'ells m the ~tro/llal cOllJpartl11cnt. DlI/mg t hl'

differentiation proce~s, the mterstltl<ll cells dcvelop a spcn:l1l/cd 1IItra~tr\ll:tllrl' ~IJJtl'l1

to steroid biosynthesls (Fawcett et aL, 19(9) and acqlllre Lli receptor:-. (Midgll'y, 1(>71;

Richards and Mldgley, 1(76) whlch are tUl1ctlcHlally couplet! to il ndl ogl'Il hlmyllt IH.':-'IS

(Erickson and Ryan, 1976; Richards and Mldgley, 1976, Fortune and Arm:-.tHllIg,

1978). Desplte the sensitivity of thc~e cells wlth Lli rcceptor:-. to pltllltary :-.tllllulatioll,

cytodifferentiation is believed tn commence mdependently 01 the pltllltary (1':~hk()1 :lllli

Luhenfeld, 1972). A deve/oping mter~tltIaI cell ha:-. a range (lI IUllctu)Jlal ()ptJ()IJ~. A

thecal cell In a developmg tol!Jcle may undergo 11Irther dIflerentlatioll t() hec<lllle all

atretic folhcular thecal œil and ~uhsequently a secondary II1ll:r~tltlétl œil (reVH~wed III

Erickson, 1985). It a folhcle dcvelops ln the point ot ovulatIon, It will undergo

luteinization and hel'ome a the cal lutem ccII of a corpu~ lutcll/ll. In il po!ycy:-.tlc Clvmy,

25

a thecal cell from a developing follicle can become an atretic follicular thecal cciI.

This cell may thcn hecome a secondary interstitial ce Il, or may possibly follow a

diflerent pathway toward hecornmg a cystic thecal cell. As ~tudies on the follicular

dynamics of developmg macrocy~tlc ovanes (Brawer et aL, 1986 and 1989) and

recovering macrocystlc ()varie~ (Convery et al., 1989) suggest that a macrocyst is an

illert structure which ~ecm~ tn he the result of an arrest of the atretlc process, it is

(llso possihle that a ey!>tlc thccal cell IS a dIrect denvatlve of a healthy tollicular thecal

ccII, rathcl than 01 an atretle thecal cell. In the microcystic ovary, mterstitlal relis may

tollow p<lthway!> simllar tn th{)~e de~crihed ahove. In light micro~copic sections of

t hese ovanes, McCart hy and Brawer (19X9) ohselved secondary mterstitml cell

c1ustcrs approxlll1atlllg the shape and ~ize ot a microcy~tic folhcle, and suggested that

SOIl1C of the !'>ccondary Iflterstltial ccII c1uster~ wlthm these ovanes may derive trom

colla p!>cd l"y~t!>.

111 con!>l(.kratloll ot the ahove, wc have asked what the morpholob'Y of these

I/ltcr!'>tiual œil populatIon!> means. How are cysts related to (lther folhcular structures?

Do ail sccondary intcr!>tltial ccli c1usters have the saille ongin? Do differences in

mOi phology retlcct drtferencc!'> m functional capacity hetween thecal cells and thecal

derivatlvl's'! If dlltcrenLe!'> in functional capacity are tound hetwecn these cells in a

l11acrocy~tlc or I11ICroCystlC ovary, l'an the ditterent functional types of thecal cells and

tlH~l"al denvatlve~ ht: related to the characteristics of the disorder (cg. gonadotropin

\l.'wls and steroid Ievels)"!

26

ProposaI

In arder ta assess differences in the structural and funl'tlonal l'harartcl isties

among thecal cells and thecal derivatives wlthin EV-induccd and E2-imI1ll'ed p()lyl'y~tie

avaries, five indices of eytodifferentiation and stcrOid synthet le ilCtlVlty will hl'

examined. The interstitléll cells under study include hcalthy ~l'romlary, atrctil'

secondary, preeystic and eystlc follicular thecal l'l'Ils, anu secondat)' inter~titlal l'db.

The indices to he examined include: alkahne phosphata sc expn:~~Hln, 1 JI hllldlllg

capacity, mitochondrial area, hrid dropJet area, and œil ~ize.

The expressIon ot alkaline phosphatase has heen Illlpllcilted tu rl'lleet

gonadotropin stimulation and stcnmlogcnic actlvlty lf1 theeal relis and Ihecal dCI iVilIIVl'~

in the ovary (MeKay et al., 1961; Pmkerton et al., 1<)61; Kenl, 1974; RO'>~, 1974, Kcnt

and Ryle, 1975). Alkaline pho~rhéltélse expressIon by the celb IJ .. lcd ahove WIll hl'

evaluated at the light microscopie level in macrocy~tlc (EV-trcilted) é1nd I1lIl/ory~tll"

(E2-implanted) ovarie~ using an Immunohistochemicalmarkcr lor alk"llIle phm)lhilt:l~l·.

Activity wlthin the different eells should retlect dittcrcrlCc~ 111 tmpie hOIIl1( Hle

stimulation and steroidogenic aetivity.

The capahility of a cell to hind LH I~ another Index 01 It~ ~en~itlvlty tll I/Ople

hormone stimulation and steroldogenic capaclty (DIl11I1l0, 1977; ))11111110 and IkflJJ:lIl,

1979; Midgley, 1973; Richard~ et al., 1976; Rowe ct al., I<JXI élml ]9X(l). 1.11 hllJlllllg

sites can he localized llsmg electron microscopIe radioélutography on OVafl:ln tl~"lIC

exposed in vivo to ellher 1251 or 11/I-hCG or LH. Both hormone!'. ~hare IllllllulIologlc

27

(Franchmont, lY70), chemical (Closset and Hennen, 1973; Bahl, 1972; Morgan and

Cantlcld, ] (71) and hiological properties (Lunenfield and Eshkol, 1967). Bath LH and

hCG hmd wlth high affinity and speciflcity to the same receptor (Lee and Ryan,

1 972h; Kammcrman et al., 1972a,h; RaJaniemi and Vanha-Perttula, 1972;

Leindcnhergcr and RClchert, 1971; Koch et al., 1974; Kammerman and Canfield,

1(72). As wcll, hoth hormoncs can he iodinated using the chloramine T method and

rdall1 thcir blological actlvity (Mldgley, 1966; Lunenfield and Eshkol, 1967; Lee and

Ryan, IY72; and Du/au, )972). As hCG is less damaged hy iodmation and is more

sté! hic (Rya n and Lec, 1(76), it was chosen as the ligand for this study. Thecal and

secondary inter~titial œil LH/hCG binding sites will be evaluated in the eeU types

h~tcd wlthin macrocystic and microcystic ovaries using electron microscopie

rad lo:tutography.

The expression of surtace LH receptors enables thecal precursors ta

ditferclllmte /rom fihrobla<;t-likc precursors to polygonal cells with the ultrastructural

char:tcten~tln 01 !'>terOld sccreting cells (reviewed in Enckson, 1985). The

lI\tra~truL'tural chafélctenstic!'> of sllch a cell incIude the prest;nce of srnooth

l'Ildoplasmic rctlculul11, mitochondria and choJesterol-containing Iipid droplets (Davies

and Broodus. I%H; Fawœtt et al., 1 (69). LH stimulation of differentiated thecal cells

and thecal dcnvatlves results In an increase in mitochondrial size (Rowe et aL, 1981),

rl'orgal1lzallon olmitochondrial cristae trom lamellar to tubulovesicular (Dimino et al.,

Il>79; Rowe et al.. 19H 1). and an increase in the ability of mitochondria ta synthesize

28

1 pregnenolone and progesterone (Dimino, 1977; Dimino Hnd Herman, 1979; Rowc ct

al., 1981; Rowe et al., 1986). Hypophysectomy results in a tl'dllction in sizl'. tlllll1hl'r

of mitochondria, and number of cristae in mitochondna. A~ weIl. the amollnt of lipid

contained within interstitial eells increases appreciahly (Carithers and Grl'L'II. 1972 il.h).

Because lipid droplets and mitochondria are ltlvolved in sterOld hiosynthl'sls and

because the size of both I~ affected hy LH, hpid dmplet areil ami Imtochondnal art'il

will be measured by morpho me tric analysis on elcctron 111ICroscoplC ladiualltograph~

of thecal and secondary interstltial cells from macrocystic and mirrocystic ovanes.

These two parameters will provide an additional index of tmpie hormone stimulation

and steroidogenic capacity wlthin different c\a~ses of thecal ccII!'. in thc~c ovanl·S.

Thecal cells and secondary interstttiai cells are oltcn classihed as "normal" or

"hypertrophied". Hypertrophied relis are consldered to he highly stemidogcnlc

(Carithers and Green, 1972a,h; Erichon, 19X5). An index 01 thecal l'l'II size will he

determined by measuring perpendicular diametcrs ot cells at the Itght IllIcrosco;,ic

level. ThiS parameter will provide an adùitlonal 1IH.It'x 111 the con~ldl'rati()1I of the

steroidogenic élctlvlty of thecal élnd secondary interstitial cells.

29

MATERIAlS AND METHODS

Animal Housing and Selection

The animais used in these experiments, Female Wistar rats (6 weeks of age,

J 75-200g hody wClght) were purchased from Charles River Ltd., St. Constant, Quebec.

They wcre mamtained under condItions of controlled light (14 haurs light/ 10 haurs

darkncss) and tcmperature (22°C) and were allowed free access ta pelleted rat food

and watcr. Fol!owing a two week accJimation period, vagmal smears were monitored

datly to c~t:lhllsh the occurrence of normal estrous cycles (Schwartz et al., 1964).

AnImais displilying at kast two consecutive four day estrous cycles were selected for

thls sludy.

Estradio\ Valcratc-Treated Animais

Thirty ammals were lightly anaesthetized with ether and given a single 2 mg

intramuscular in je cL on of estradiol valerate (EV) dissolved in .2 ml sesame ail (Squibb

Canada, Ine, Montreal, Qucbec) in order tn induce peo (Brawer et al., 1978). Since

Il hll~ hecn weI! t'~tahltshed tllat an injection of sesame Oll alone does not alter either

the morphology or fUI1ction of the hypotha\amo-hypophyseal-gonadal axis (Brawer et

i11.. Iln~ and IW,{); Farookhl et al.. 1985; Hemmmgs et al., 1983, Schulster et al., 1984;

SlIl1l1ld et al.. 191'7), sesame oll-II1Jected animaIs were not prepared as contro]s for

tllls ~tlldy. Although cystlC follicles are present withlll the ovary twenty- eight days

tollowing II1JèCtlOI1 nt e~tradlol valerate (Brawer et al., 1986 and 1989), a period of

ftItY-SI\ days was allowed to elapse after injection as cysts are fully expressed by this

30

time and the morphology pf the ovary l'rom this time point on reJll<lins constant , ,~

(Brawer et al., 1986; Schulster et al., 19R4). Eight weeks alter EV-trt'atment, tlH1Sl'

animaIs which demonstrated unequivocal and sustained cOfl11l1ed vagmal smears dut ing

the two prior weeks were chosen tor fmther study.

Animais Receiving Estradiol-Containing Implants

An additional twenty é1nimab were lightly :tnacslhetized with elher and

Polydimethylsiloxane (Silastic) capsulcs containlllg 17-1~-estra(lIol were tl11plantcd

suhcutaneollsly. The ~lIastic capsules wete prepared accordlllg 10 the nH.'lhod 01

Stratton et al. (1973) u~lI1g a 2 ml11 long piel'c 01 polydllllcthylsiloxalll' tuhlllg wlth an

outer diameter of 3. J 8 mm and an inner dlamcter of \.9X Illlll (no. 602-JO\ Dow

Corning, Midland, MI). The release nlte 01 thesc capsules was apploximatc\y

2.4j.Lglcmday (Rohaire ct al., 1979 and 19H2). They have heen ~hown lo mamtalll a

constant pla~ma estradlOl concentratIon 01 approxllllatcly 50 pg/ml (Brawer et al.,

1983). Six weeks after placement of the implants, vaglllai ~l1lcan, werc mOllllorcd

daily. Ali a 111 mals used in this study were in a stale of ~tlstaincd vag 111 a 1 corllllicatlon

hetween the sixth and eighth week altcr implant placemcnt. Thl~ II1tcrval wa!'> ~c\ccted

as the polycy~tic morphology 01 the ovary I~ lully exprc!'>!'>cd by elght weeb aller

placement of the implant (Dr. J.R. Brawer, unpllhlJ~hcd oh!'>crvatlon).

Experimental Procedure M Alkaline Phosphatasc Sludy

Eight weeks aiter either EV treatment or ~I1a:-.tlc cap:-.ule Implantation, two

groups of eight animaIs, each dem()n~tréltll1g per:-'I~tent vaginal cornillcatioll uunng the

31

•

prim cight uays, wcrc sclected for this study. As weil, eight untreated, cycling, age­

matchcd (,~mtrols maintained under identical housing conditions as the hormonally

treated ammals were used. The animaIs were sacrificcd by decapitation and their

ovaries were removeu, decapsulated, weighed, and snap frozen in liquid isopentane

(Sigma, St. Louis, MO) at -40°C. The avaries were then stored in a biofreezer

(-90°C) until section cd.

Frozen whole ovaries were mounted on a chuck using a.c.T. compound

(Tissue-Tek, Miles SClentiflc) and scctioned at 20~m intervals on a Reichert cryotome

maintaineu al -zone. The sections werc thaw-mounted on gelatin-coated glass slides

and thcn storcd at -<JO°C untJ\ Ill1lmmm.tained for al kali ne phosphatase activity.

Pnor ln stammg, the sections were removed from the biofreezer and air dried

for twcnty Illll1ulcs al mOI11 temperature. They were then washed in cold phosphate

hult'ercu saline (PHS) and suhsequently fixed in absolute ethanol at 4°C for thirty

scconds (Ponder and Wil klll~()n, IWO). For the immunohistochemical demonstration

of alkalinc phosphatase activity, the standard ABC Vectastain rnethod using antibodies

against alkahnc phosphatase H (Vector laboratories, Burlingame, California) was

employcd. TI~SllL' sections were Illcubated in a hurnidIfied chamber in a solution

cOlltamlllg 1 abhlt anti-alkahne phosphatase H antlbodlr~, biotmylated goat anti-rabbit

I1l11l1UIlOg1obu11ll molccll1e~. anù an avidin-biotin complex cou pIed with phosphatases.

The llptllllal IIlCUhatlon time was determined by incubating sections for varymg

:Imounts of time hetween filteen and thlrty minutes. The best incubation time was

32

found to be twenty-three minutes, as incubation times longer than this rl'sultcd only

in an increased intensity of the reaction with no variation in localization 01 the

reaction.

Following a twenty-three minute incuhation periml in the ABC-AP suhsttatl'

solutions, the sections were washed for rive minutes in several changes 01 glass-dl~tilkd

water, counterstained with toluidine hlue, air dricd and coversltpped.

Control sections were ohtained hy inclIhating OVanéll1 tlSSUC ~ections in the

substrate solution prepared without the primary antihody. Background staining, wlten

observed, was very low.

Alkaline phosphatase expression was assessed in ail classes 01 folhcles (il'.

primary, secondary, Graafian, precystlc and cystic), ln emportl I\ltca and 111 the rclls

of the stromal compartment. Immunoreactivlty lor alkaltne phosphatasc was CVillllatl'd

by vis liaI inspection at the light mH.:roscopic Ievel. A ratmg M.'ale will he lIscd III the

text and in a summary chart. The 1I1lIts which WIll he lI~ed ln indcx :lIkallllc

phosphatase expression incIude: "0", "+", "(+)", "+" and "++". "0" dCl10tcs no

activity, "+" an occasional positive reactinn, "( +)" a weak reaclion; "+" a conmlcnt

and definitive positive reactlon, and "+ +" a ~tr()ng ïeactIOJ1. Bcc:lllsC va~clllélr

endothelial cells of the rat ovary ~how considerable alkahnc plHlsphélta~e actlvlty and

may, therefore, cause occa~ional diffusion artlfacts Icading to the ~tall1l1lg 01 \truetllrc:-.

which may not be specialized with regard to alkahnc ph()~phalél~c actlvity, an mlemk

is used ta sigmfy when at least part ot the reactIOn ()h~ervcd for a partlcular :-.trllcture

33

is given by hlond vessels.

Experimental Procedure - hCG Study

Preparation and Administration of hCG Radioligand

Eight weeks after EV -treatment and capsule implantation, two groups of nîne

animaIs each were anaesthetized by an intraperitoneal injection of 15% urethane

(Ethyl Carhonate) at a dose of 1ml/100g body weight. Subsequently, each animal

received an intfélJugular injection containing approximately 100 x 106 cpm of 125I_hCG

dilllted in 0.2 ml of PBS. The quantity of radioactivity used was based on prevlous

stlldics in our lahoratory which indlcated that thls dosage results in a level of

rat!Jo(Jctlvlty withlll the ovary which IS lOO-700x in excess of background levels and,

consequently, IS sulhcient to provide a good representation of the numher and location

of LH hinding sites present withm the ovary (M. Richard, 1988 and Brawer et al.,

19R9).

The iodinatlOn of hCG was performed using the Chloramme T method as

dcscrihcd hy Ircland and RIchards (1978). Highly purified human chorionic

gonadotropm (hCG) (CR-12], 13,450 lU/mg) (Morgan et al., 1974) used as the

jodinatcd ligand WélS éI gin t'rom the Center for Population Research of the NICHDD

of the National Health Institute (NIH). The specifie actlvity of the 125I_hCG prepared

for tlm study was approxlI11:ltely 50 J.LCi/p.g for the EV-treated animaIs and

appmxlIllatcly 45 J.Lel/J.Lg for those élflllllals chromeally exposed to estradiol via. a

sllastic Implant.

34

As a control for the specificity of the radiolahelled ligand. thrce EV-inJl'l'tl'd

animais and three Erimplanted animais were injected wlth the saml' dosl' 01 12'1_

hCG along with a thousand fold excess of unlahelled hCG (Ayerst LahO! atmy.

Montreal, Quebec). Lahelling ohserved 111 the ovaries of tllt.'sl' :1 III 111:11s altl'I

radioautographic processing would represent non-specifie l.lbc1hllg.

A period of thirty minutes was allowed to c1apsl' alter II1Jcctio\l 01 the

radiolabelled ligand before sacrificing the animah:. This exposure t1lllC was seketl'd

based on findings of Rajeniemi et al. (1974) and Han et al. (1<)74) whICh mdICatl's that

there is no difference In the level 01 lahellmg in the ovary at live or ~ixty Illllllltl'S post

125I_hCG administration.

Animais were sacrillccd hy pertu~ion VJ<l. the abdolllmai (Jort:!, arcordlIIg tll thl'

procedure described by VItale et al. (1973). The abdol1llll:t1 cavlty was expmed

through a midline abdominal inciSIon anù the H1te~tll1es were retracted III Illdl'I III

expose both the ascending vena cava and the ahdomlnal :torta. Alter the IWIl vl'!>.~eb

were separated, the descending aorta was cJamped Illllllcdlatcly "hove Ihl' 111:1('

bifurcation. An 18 gauge needle was introdllccd mto the :lOrta, ahllve the damp, ami

perfusion wlth lactated Ringer's ~()llIti()n wa., heglln. Upon clearance 01 hlood IIOIIl

the kidneys, a second clamp wa~ placed JlI~t ahllve the renal velll, an II1CI~I()11 Wil~

made in the right rena) velI1, and the rerlll~ate ~()llItl<)1l wa,> ,>wltched tn il O.12M

phosphate butfered 1 % lormaldehyde - 1 % glutl.:raldchyde ~()llItHlIl (Jf pli 7.4

(Karnovsky, ] 9(5). ApPf()XJmately 150 ml 01 IIxative wa,> perlll~ed through the ~y~tem.

35

Fol1owing perfu~ion, the ovaries were removed, deeapsulated, weighed, plaeed in a

gamma counter to determme the quantity of radioaetivity taken up, and finally placed

in fixative overnight.

Preparation of lhsues for Radioautography

The next morning, the ovaries were eut into two pieces each and then washed

for five mmute~ in a cold solution eOlltainmg dextlmc, 0.4% standard phosphate

huffer, élnd calcium chlondc. They were then post-fixed in a solution of 2% osmium

tctrclXldc élnd 2% potassium fcrwcyanide for two hours (Karnovsky, 1971). Following

post-fixation, the ovanes were washed tWICC in a 2.4% calcium chloride solution,

dehydratcd III a graded methanol sene~, and embedded in epon.

Pale gold !\CCtl\H1S ot 50-10011111 were cut from epon blocks using a diamond

knife and a RClchert microtollle. The !\ectlons were mounted on Athen-type EM

copper gmls (JOO Illcsh) u!\ing an asplrcltor with attached flIter paper. Within seven

days lollow1I1g thl' II1Jcction of the radlolahelled hCG and perfusion of the animaIs, the

ovarian tissue sections were proces~ed for EM radioautography.

Electron Microscopie Radlo:lutography

The sectlon-hearmg gnd~ were coated with Jlford L4 Emulsion (Ilford Photo

Cana la. Markha 111, Ontano), exposed for 6 months (exposure time was based on the

results of ~ectioI1!\ lrom the saille ovaries whlch were processed for light microscopic

radloilutography) and then developed for fine grain electron ITIlcroscopic

radlOillItography hy the method of Kopriwa (1975). The sections on the grids were

36

then stained with a solution of 4% uranyl acetate \Il I11cthanol for 5 minutes, nn~cd

with glass-distilled water, and eounterstaincd with 0 1 % Icali citratt' fOI two minutes.

Electron Microscopy

Electron microscopie examination of thc ovanes was rarrieli out on l'Ithcr a

Siemens 101 or a Phlllips 400T electron microscope. Thccal cells \wrc localin'd to

healthy follic1es, atretle folhcles, precystlc and cystic tolhc\c~. As weil, ~l'rolldary

interstitial cells were localized. Ten thecal cell~ each lrom hcaIthy scrol1dary lollIcks,

atretic secondary folliclcs, preeystlc folhc1cs (EV -trl'ated ovanes only), ry~t Ir l{llhrll's

and ten seeondary interstitléll cells [rom both EV -trcatet! and ETtl"eatcd oval Il'~ weIl'

photographed without bias at a magmhcatlOn ot .5, ()()()x. Thll~, 1 III ty theral rl'lb Ir (lm

macrocystic ovanes, thlrty thecal celb lrom Il1lCroCystlC ovalll'~, ami tell sl'l'olldary

interstitial cells from macrocystic and mlcrocystlc ovancs, were analy/ed. For carh rdl

type examined, at least one photograph wa:-. taken lrom earh 01 the SIX EV-treatl'd

animaIs and each ot the E2-lmplanted animaIs. In other wonb, Iwo phot()gl<lph!-. Wl'Ie

taken from the structures wlthin the ovane:-. 01 tour dtlterellt élnllllah wlth l'Ithl'f a

macrocystic or a mlCrocy~tlc condition, and one photograph wa') takcll tWill the

structures wlthm the ovanes ot two dlttcrcnt anH11;I\!-. wlth elther a Illacr()cy!-.tlc or a

microcystic ovarian conditIon.

Electron mlcrograph:-. ot thecai and !-.ccondary IIlter:-.tltlal cciI!-. were exallJllled

for change~ 111 the size of brid droplet:-. and ITIltochondmd area wlthlll the cyto')()1 III

the various classes of celI~. Lipid droplet area and mitochondnal area were Illea!-.urcd

37

with a MicroPlan Il image analyzer. Radioautographic grains in each cell were counted

trom the electron micrographs, according to the guidelines established by Kopriwa and

Nadler (19R4) for fine r~ain, solution physlcal development of electron microscopic

radioélulographs with 1251 as the isotope. Because of the size variatIons between

thccal amI sccondary interstltial eells ot the same origin and between the different

elas~c~ ot ccII!., grain c(}unt~ were expressed as per 1,OOO,um ceJl surface length as the

receptor IS located on the pla~ma membrane (Rajaniemi and Vanha Perttula, 1972;

Raj<lfllclllI et al., 1974). The ~urtace length ot the œIl surface was measured by

traclllg the plasma memhrane lIsmg a MieroPlan II Image analyzer.

DeterminatIOn ot CcII Size

The ccII size for each type of thecal cell and for seeondary interstitial eeJls was

mcasured lIslllg a hght mlero~cope with an ocular micrometer. Measurements were

made at a magl1IlIcation ot 2,OOOX. Perpendicular diameters of four cells each from

healt hy secondary t()lIide~, atretle secondary follicIes, precystic folhcles, cystie follicles

and secondary IIlter~tltral cell~ t'rom flve dlfferent ovaries were taken. Measurements

Wl'fe made <Hl cell~ whlch were cut through the plane where the nueIeolu~ ot the cell

was vIsible. The average ot these two measurements wa~ then calculated to provide

an IIldex ot œil afl~a~ lm theral cells from healthy tollicIes, atretie t'ollides, precystie

and cy~til' tollrcle~, and ~eCl)ndary interstitial cells.

38

Analysis of Data

Mean cell size of thecal ce Ils l'rom healthy follic1es, atretic follides, prccystic

follicles (macrocystic condition only), cystlc follicles and secondary intclstItialcclls hOIl1

both macrocystic and microcystic ovaries were comparcd llsmg Stlldellt's t-test.

Differences were considered signiflcant at p<0.05.

The mean lipid droplet area tor each ccII typc cxal11l11cd (ie. healthy SCCllIH.l:lly

follicular theca, atretie secondary folhcu lar thcea, preeyst ie t hee:! [maerocyst le l'Olllllt Il J\l

only], cystic theca, and ~econùary II1ter~titial relis) wa~ ealclllated hy dlvllllng the total

Iipid droplet area trom the ten relis in that eé:ltegory hy the total III 1 111 I)L'I !lI Itpld

droplets scored in those ten cells. Mean hpid droplet areêt hlst!lgral1l~ weIl' gCllclatl'd

for both macrocystic and microcy~tic ovarics. In addltloll, the pClccntagc 01 the

cytoplasmic area of the œil profIle occupied hy Iipld wa~ dctcrl1l1111'd. The cytopla:-'l1llc

area of the cell profIle was ealculated hy sllhtracting the nuclear alea Iwm the total

cell profile area. Nuc1ear and cell profIle area:-. werc mea~ured lISlllg a MlcroPlan Il

image analyzer. Multl-group comparison~ of the data trom l'élch type ot (lv:lly WCll'

analyzed by one way analysis ot vanance. A compan:-,oll ot IllCéll1:-' hctwccl1 the

different cla~~e~ ot theral or ~econdary inter~tltlal group:-. trom m;H.:rocy:-.tlc ()f

microcystlc ovanes wa~ made lJ~ing a Studcnt\ t-tt:~t. Dllfcrcncc:-. werc com.ldercd

significant at p<().O~.

Mean area of mitochondnal profile~ for each cell type cxamincd wa~ calculatcd

by dividing the total mitochondnal area for the ten ccll~ III that category hy the

39

numher 01 mitochondrial profiles scored in those ten cells. Histograms of mean

mitochondrial area were generated for both macrocystic and microcystic ovaries. The

pcrccntage of the cytoplasmic area of the cell profile occupied hy mitochondrial

proliles WélS again determined. Multi-group comparisons of the data from each type

01 ovary wcre analyzed hy one way analysis of variance. A comparison of means

hctween the diffcrent classes of thecal or secondary interstitial ccII groups from

macrocystic or microcy~tlc ovaries was made using a Student's t-test. Differences were

considercd slgnilicant at p<O.05.

40

~------------

RESULTS

Histological Features of Po/ycystic Ovaries

Light Microscopie Description of Macrocystic Ovarles

In the estradiol valerate-induced polycystic ovary, primonhal and plllllary

follicular populations appear as they dl) m healthy, control OVé\ncs. The 1l11lllhl'l 01

secondary follicJes contained within the macrocystlc ovafle~ IS, howcvcr, dlllllnishl'd.

Most of the secondary follicJes are atretic, and thcy arc unllsually ~l11al1 111 SIZl'.

Graafian follicles and corpora lutea are ahsent t'rom thc~e ()vane~.

EV-induced polycystic ovaries conta in thrcc hlstologlcally distlllet large lolllclIlar

structures which are not oh::-.erved ln untrcated, normally cycJing ()Véllll'~. The III st, t hl'

macrocyst, is a large structure characterlzcd by an attelluated IllCmhrallil gralllllo\a

(one, or occaslonally up to three celllayers tlm:k) and a hypcrtroplm:d theCétlcl'lll:IYl'l

(Figure 1). Interming\ed wlth the Iusltorm granlilosa ccll~, or Irce III the illltl al ~pacl',

are large macrophage-like cells containing den~e inclu~i()n granule .. and lrothy, gho~t­

like droplets. The hasement memhrane surroumhng the Illcmhrana gralllll()~a I~

thicker than ln healthy developmg 1()llIclc~ or Graallan f()llJclc~ 01 a cycJlIlg ovary.

The hypertrophied thecal cell layer con!\ists of large, polygonal cells whlch me dCI1!\ely

packed with hpld drop\et gho!\ts, glving them the appearancc (JI bClIlg lutl'llllzed.

Their appearance is dl~tlnct from thecal celb of hcalthy f()lhclc\, wlllch éln: \l11all,

fusiform in shape, and contam much le~!\ IIpld. II()wever, thcy are ~tnklllgly ~llllIl;lr

41

in appearance to secondary interstitial cell c1usters found scattered throughout the

stroma.

The second type of large follicular structure is characterized by a large antrum

lined by several irregular layers of granulosa eeUs in different stages of degeneration

(Figure 1). The granulosa cells are polygonal in shape. Phagocytic cells are only

occasionally seen interspersed between them. The tIlecal ce]) layer is hypertrophied,

as IS that ot a cystic follic!e. However, smaH fusiform cells are intermingled with the

lipid-tïlled, hypertrophied polygondl. cells. This structure is referred to as a precystic

tollicle (Brawer et aL, 1lJH6, 1989; Richard, 1989).

The t1md type ot large follicular structure unique to an EV-induced polycystic

ovary 15 the Type III Large Folhcu!ar Structure (Figure 2). This structure is larger

than prcovubtory tolhde!.. It consists Dt a thick membrana granulos<l, an undulatt'd

hascmcl1t membrane, and;.: diffu . .;e layer of ~,mall, fusiform thecc.ll celIs with occasion al,

scattcn:d large r"olyg(1J1al ce Il!>. The granulosél cdls of thl5 follicle differ from those

of a hcalthy secondary follicle in that they arc smaller, more variable in shape, and

more dCllsdy packed. In addition. mitotic figures élIe observed In the peri mural region

ot the J1lcmhrana granulosa. The thecétllayer of a Type III Large Follieular Structure

dllfciS twm that Dt a healthy developing follicle in that it has a festooned appearance.

Grossly. thb ~trllctlJl"e dllteïs from preovulatory follicIes in that it is larger and contains

110 ov li 111.

42

Secondary interstitial cell c1usters ar~ a normal component of the ovariun

stroma, generally thought to be derived from collapst!d atrellc folljdes (Kingsh\JlV.

1939; Dawson and McCahe, 1951; Greenwald, 1968; and Deancsly, 1'172). They ait'

unusuaHy abundant in these polyeysttc ovaries. The~c polygonal cells an' laI gt.' a III 1

are filled with lipid ghosts (Figure 3).

Five classes of interstitial celis from these macrocystic nvarics will hl' stuùied'

healthy and atretic secondary follieular thecal ce Ils, preeystle and eystic thccal l'Clis,

and secondary interstitial cells.

Light Microscopie DeSCrIption of Microcystic Ovaries

The histological differences betwecn ovarics continllotJ~ly cxposcd to

physiologlcal levels of estradiol for eight wceks and normal, control ()Vlllll'S élit'

striking. However, hke EV-treated ovaries, Erexposed ovanc~ dn not cnntlllll corpma

lutea. As well, large secondaly lollicles are rarely observcd, and those that arc, ale

nonnally atretic.

In the cortical region of the ovary, numerolls small eystlc f()lIicle~ ale prc~elll

(Figure 4). These cystic follicles are cOl1siderahly smaller than thelr cOlllltcrparts III

a microcystic ovary (average dmmeter 740jLm ver~lIs 229JLm, rcspectlvcly 111= lOI). The

membrana granulosa of these cystie follicle~; conslst~ of bctwecn onc and four layer~

of degenerating cells and an unusually thlck layer ot hypcn <'phlcd thecal ('db. Thc

the cal cell layer is apPf(lXImately four times the t}1J( .. kncss 01 ttlélt 01 the lllilCrocySt.

At the light microscopie level, the cytoplasm of the:-.c thecal ccl\<' appear~ to I)e

43

entirely occupied hy lipid droplets.

The mterfolhcular (stromal) area of the microcystic ovary is very distinct in its

appcarance (FIgure 4). Nurnerous extensive clusters of secondary interstitial cells are

scattcrcd throughout the stromal mea. The cel1s within these clusters are large, pale

staining and frothy. The frothy appearance of these cells retlects the fact that they

are cngorgcd wlth hpld ghosts. The sizt' and shape of sorne of these cell clusters

approxil1late~ that of a cystlc folbcIe, suggesting that they l1lay derive from collapsed

cysts.

Four classes 01 cells lrom these mierocystic ovaries were studied: healthy and

alrelie secondary fnllieular theea, cystic the cal eells, and secondary interstitial cf'lls.

ALKALINE PHOSPHAT ASE

1\ SlIl1ll11ary of the distnbutton of the immunohistoehemical marker for alkaline

phosphatase wlthlll the variolls cells types of avaries treated with either a single

IIlJcction 01 estradiol valerate or exposed to chrome physiologieal leve]s of 17-{3-

estl mhol vIa a sllas!ll: Implant, along with the distributIon in untreated control avaries,

IS plcsentcd III Tahlc 1.

Alkalll1l' Pho~phatasc: Primordial and Primary Follic1es

In ail three types of ovaries studied, no alkaline phosphatase immunoactivity

(0) was ohscl"ed Jl1 the memhrana granulosa of follicles at any stage of development,

whether healthv or atrelle.

44

Alkaline phosphatase expression in Hie stromal cl'lls cndrding tht' primordial

foIlicIe, which have not yet differentiated mto thecal cl'lIs, is very wcak (±) and

infrequently observed. In marked contra~;t, a positive (+) alkahnc plh1"pha!asl'

reaction is observed consistently in the cytoplasm of the dcvcloping cells of the thcl'a

interna of primary follicles (Figure 5).

Alkaline Phosphatase: Secondary and Gnultian Folllcles

A strong, positive reaction (++) fot alkaline phosphata~c i~ ohscrved in l'clis

of the theca interna of healthy sl'condary tollic\es withm the ovarics of control, EV­

treated and E2-implanted animais. Graafian lol1ic\es, present 1l111y 111 controlllv:tl il's,

also dernonstrate a strong positive reactlOIl. Alkalllle pho~ph;.'t:tsl' CXPITS~I()n 1 ...

maximal at this stage of follicular development, as the wa ct Il III p' oduct oh:-.c, vell Ill' Il'

following immunostaining IS the stronge~t one observcd lm ally eclls 111 the ()vary

(Figure 6), Staining is ohserved dittusely withm the cyt(}pla~1l1 01 thl'~C ce Ils, wlth

sorne cells showing a weil defined peripheral cytopla~l11lc zone whlch ,~ m:hcr III

al kali ne phosph:1tase content. As weil, the al1lOlIllt 01 alkalllle ph()~phata~l' actlvlty III

these eells increase~ as one /l1Ovl'S outward t'rom the ha~cJ11enl II1cmhlélnc.

In addition to the ~taining localized to the cell~ of the thcca IIItcrna 01 thC:~l:

follic\es, endothehal cens 01 thecal cap:'!driC'i abo ~h()w a ~tf()/1g, P()~llIVC rCilctloll

(+ +) followmg immunostaining (Figure 7).

45

Alkaline Phosphatase: Atrctic FollicIes

The theca interna of atretic antral follicles displays a strong, positive reaction

( + +) for alkahne phmphatase, regardless of the degree of atresia expressed and of

the type of ovary fr<Jm which the section was taken (Figure 8). The density of the

reélction product i~ comparahle to that observed in a non-atretic counterpart.

Alkalmc Ph(}5phatél~e: Corpora Lutea

Corpora lutca arc ohscrved only in the control ovaries (Figures 6 and 8).

Becausc the ~CctIOIl~ arc prcparecl from frozen ovaries, resolutioll of morphological

detmls 01 the œlb IS lI1adcquate to permit classification ot theca) and grnnu)osa lutein

ccll~. Staining ot cdb wlthin these structures is hlghly vanable. The cells of corpara

lutea wluch acculllulate rcaction product tollowmg incubation with the enzyme marker

arc deèply st,lIllcd (+ +). 1 n ~ome cases, almost ail of the cells comprising the corpus

lutelll11 arc ~tained. In other ca~es, staining is restricted 10 ce:ls near the central

coagulum or to radial stramls 01 cell~.

Aikallllc PllOsphata~e: Secnlldary Interstitléll Cells

The 1111111111:0St:11l1lIlg 01 ~econdary interstitIaI cell!. 15 highly variable hetween the

control. EV -tlcated and ET lll1planted ovary. In control ovaries, immunostaining

OCClIlS pnlll,lfIly over vasl'ulm endothelml ce Ils. SIllaIl c1u~ters cO.1sü,ting of ten or

k\,,~r Illter!\tl1li1l celb show a T/loderate (+) reaction to the alkaline phosphatase

alltlbody rompkx, but these arc inlrequently ob!ierved III the interfolhcular spaces and

seldol11 tOll!1d 111 the hllar region of the ovary (Figure 9).

46

In the estradiol-valerate treated ovary, a strong positIve rractlon tOI' alkaltne

phosphatase is most prominent amongst vascular endothrltal Cl'IIS. F:unt stain1l1g

occurs over intermittent c1usters ot twenty or s() interstltlill n~lIs (+) located holh

peripherally in the mterfollicular spaces and 111 the reglon 01 the 11Illis. 'l'hl'

occurrence of these alkaline phosphata se positive œil c\UStCIS IS IlltiLh mOlc hl.'qul'nt

than in control (11f~althy) ovaries, hut the staining is ~uhstalltially wcak.'1.

In cystic ovaries mduced by ehronÏl: exposure to 17-t'-cstradlol VIii ~Ih.tll'

implants, small strands ot seeondary mterstitlal eells ~howlllg li strollg P()~IIIVl' 11.'actU)1l

(+ +) for alkaline phosphata~e are ohserveù in the penphcral and 11Ixta-htlill rl"gHllls

of the ovary. The mtensity of the stalllll1g of the~e cell~ IS !'>lIl11li1r to Ihal ()h~l.'lvcd

within the va~cl1lar endothelial celb. Ocl'a~i()nal 11Ighly rt'ilctlve (-+ +) ~/l1all. roulld

cJuster~ of celb élre oh~erved in the peripheral regHHl!'> 01 the cortex (FlgUlc 10) III

addition 10 these highly rl'active strand~ and c1u ... ten, oj l'l'lb, .... malln rlu!'>ll.'l!'> 01

interslitiaJ rells, ~il11ilar 111 slze tn those wlthin an EV-treated (}vary and 1:llIItly \t:l\lled

hy reaction product (+) are ohserved dlspersed throughollt the IIltcrl()lIll'ular al e:l~ ()I

the cortex.

Alkaline Phosphata~e: Precy~tle Follicles

Precystll' follicles are encollntered ol1Jy wlthin e~tradl()l-vall'rate treated cy~tlc

ovaries. They are dl~tingUl~heù lrom true cy~tlc 1()lIlcJl'~ hy the pre!'>enœ of Irregulal

and often rather extemlve patche~ of ùegeneratll1g gralllllo~a cclb alollg Ille <l1111;t!

surface of the memhrana granulo~a. A n10ùerate ~tamlllg reactlon (+ ), ~ll1ltl:1l tu 111;11

47

ohservcd over the thecal wreath of primary follicles, but weaker than that observed

over thecal cells of antral follic1es, is observed over these thecal cells. The vascular

cndothelial eclls are éI bit more prominent here as they stain more intensely than the

hackground ~tainJng of the ecll~ of the theca interna. The membrana granulosa of

thcse follicles rcmams negatively ~tained (0) (Figure 11).

Alkalme Phosphatase: Cystlc FollJcle~

Cy~tlc t()JlJcle~ arc oh~ervcd ln hoth EV-treated and Erimplanted avanes, but

are ah~ent lrom control ovanes. In hoth types of ovarie~, the attenuated granulasa

ccII layer i~ stamcù only with tolUidine hllle (0). The hypertrophied thecal layer

characteri~tlc ot the~e cystIC lollicles IS very weakly stained (+) In comparison to the

stailllng oh:-.crved on either pnmary or antral tolhc1e~, healthy or atretle il} both the

l1lacrocy~tlc (Figure 7) and microcy~tlc (FIgure 12) condJtlon~. As weil in rnost of the

cystic folhcle!'>, ol1ly the two layer~ ot thecal eclls adjacent to the hase ment membrane

me st,lIllcd hy the ellzyme marker. In no mstance i:-. the thecal ring totally unreactive.

The l'm.lothclwl cell~ wlthll1 tl1ls layer of relis remain strongly reactlve (+ +).

('oll:-.cqul'Illly. Il 1:-' possihk that :-.ome of the stammg i:- due to dlfluslOn ot the reaetive

pllldUcl lJom the \'a~ClIlar sy~telll.

48

ELECTRON MICROSCOPIC CYTOLOGY OF THECAL AND SECONDARY INTERSTITIAL CELLS

Estradiol Va/erate Treated Animais - Macrocystic Ovarian Condition

Light Microscopie Analysis of Cell Size

A mean index of cell size ot theeal ('t'Ils from healthy and atrrtll" sl'l"Ol1llary

follicles, precystic and cystic follicles, and of !>econdary interstltml cl'lIs, IS SUll1l1lall/l'd

in Figure 13.

The mean cell size of healthy secondary folliclIlar theeal l'clis + standard t'I ror

of the mean (S.E.M.) is 9.R + .4J-t. Thelr size IS signllteantly ~l1lalll'r than tht' Illl':tll

cell size of the other tour cell types examineù (p < .0(1). Mean éttlcttr sccondary

follicular thecal celI size is 12.0 + .3J.L. Mean ccII ~Ile ot plery~tlc and l"y~tll" thcl"éll

cells is 11.2 + .3J-t anù 11.R + l.Oj.L, respectively. Cy~tle theral cciI!-. arc ~lgllllIl"alltly

larger th,1O precystlc thecal cel1s. Seconùary inter~tltial ccII ~ll"e 15 Il.3 + .3J.L.

Electron Microscopie Iùentiflcatlon 01 Thecal Cell"

Three ùitferellt proecùure~ were lI~eù tn local1ze thecal eclls 10 healthy

seconùary f()lhc\e~. atretic ~econdary 1()IIIc\e~, precy~tlt' lollJr\e~ 01 cy~tlr 1()1I1c\l"~

First. a I1ght IllICrmCOplC ~hde cOlltallllllg the ~al11e 'ectloll a:-. thl' t'lect/()/1

microscoplc gnd wa~ ~tllùleù tu lùentlly the lolllclllar ~trllctu/e, pre~ellt O/J the gml.

Seconùly, at the electron IllICrmCOplc level, the grélllulmél cell~ 01 the 1()IIIc\e

in question were examineù. The followmg characteri..,tlc!'> provlded the enterré! lor

identification of the follicular ~trllcture hcrng examlllcd.

49

1. Granulosa cells of healthy secondary follicles are polyhedral In shape, and

contain triangulctr or oval nudel, evenly distributed ribosomes, oval mitochondria with

lamellar cri~tae, smooth and rough endoplasmlc reticulllm, and Golgi complexes within

their cytoplasm. The hasal layer of granuiosa cells are low colllmnar ln appearance.

Ali of the cells of the membrana granulo~a are c10sely apposed. The underlying

basement memhrane appean. Intact.

Il. Granulma cell~ 01 atretic ~econdary follicles are often separated l'rom

each olher, and arc lIregllJar 111 ~hape. Thl~ i~ e~pecially 11 ue of the periantral cells.

N uclcl are elther oval or triangular, and elther basally or centrally located. Often, the

()rganl'lIc~ are c1l1111ped ilround the nucleus, or al one pole ot the cell. Cytopla~mic

h\chblllg I~ oltcn ()b~crvcd ovcr the apIcal membrane of periantral cells. As weil, lipld

droplcts Ilormally Sl'cn only in the ha~al layer!; of the membrana granulosa of well

l\cveloped secondary l(JlIlc\e~ (Pe\u~o et aL, 19XO), are ~een in scattered cells at ail

leveb 01 the memhrana. The ha~cment memhrane ot thc!le folltcles appcars normal.

III. The I11cmhrillla granlllll~a of precy~tlc follicle~ 1), very dl),tmct. lb thickne~s

I~ 11Ighly Irregular. The cells arc variable tri shape. and cell bOllndane~ ohen can not

Ill' dl~tlnglll~ltcd. Nllclear ~hape is abo highly Irregular. Small mdentatlons of the

Ilucll'us, Ilot ~et:1l in gralllliosa cells 01 healthy or éltretlc follicles, are observed.

Cytoplasl11lC bkhbmg IS agam ohserved over the apical memhrane nt scattered c1l1sters

01 pl'nalltral gralllliosa ce Ils. Organelles appear to he scattered throughout the

cytoplasl11 ot tItCl-l' l'db. In contrast to atretlc fnlhc1es, lipid droplets are observed

50

only in sorne of the granulosa cells. and almnst exclllsively wlthin l'clis of thl' hasal

layer. In addition, numerous alltophaglc vacuoles are OhSCrvl'd in granuillsa l'l'Ils 01

these precystic fnllicle~. These are not ohserved Hl the I11l'mbrana gr.11I1110~a 01 ally

other follicle type studied (Figure 14).

IV. Three features of the mcmhrana granlllosa of il cy~t th!.tlllglll~h titis

follicular type from the other tluee. Flr~t, it is the only ra~c wherl' the IllcllIhlall:l

granulosa consists ot only a smgle layer of ce Ils. Secolltlly, the 1I11dl'i 01 thl'sl' l'db

are highly indented and irregular in shape, althollgh the chro1l1atlll patlnll I~ 1101

disrupted. T/md!y, large, Iffegular IIIterccliular ~paces arc IIequl'lltly oh!'>t'rved

hetween the areas fu!>ed ny Junctional comp/exc!> of two adjilcl'Ilt gr;l/llI/o"';1 n'lb.

Occasionally, cytoplmrlllc extenslom. ot two adjacent grallulosa l'l'lb pn)ject 11110 thl'!ll'

spaces (Frgure 15). The nasa/ lamJ1lél lImlerlying the cy~tic 1I1emhralla gralllll()~a

appears hypertrophied 111 some arcas, disruptcd in ~oll1e area~, alld rl'glliar III (lther!..

The final critcna u~ecJ to localIlC thecal ccII ... \() a P;lI tlcular jollll'ttlar type

considers the morphologlcal charé\ctemtlc~ ot the thcc.1I l'l'Ii rt!'>elt.

Electron M Icrmeoplc Dc~cnptl()n <lI Thcc;t1/Secondmy Il1ler!.t I)J.uL _LdJ Morphology

The tlleca interna of healthy :-,ccolldary follJcb contalll f lI~lIorlll-~llapeti

steroidogenic cells Iflterspersed hetween ~pll1dle- ... haped tJhf()hl!l~t~ «lId c;I(Jlllary

endothehal celh.. The~e cell~ contéllfl :-.phcflcal r11ltochofl(lrr:t wrth tuhular or ve\/cldélr

cri~tae, extensive ~m()oth endopla\mic rt;t/culum and IIp,d drop/ct. .. (hgurc 1 (»). The

thecal celb turther t'rom t:le hH:-.ement memhrane tend to contaI/) J11uch more IIp/d.

51

Nuclei are generally euchromatic, although the chromatin pattern in sorne nuclei is

hctcrochromatic.

Thccal celb l'rom atretlc secondary follicles are morphologically similar to those

of il hcalthy ~econdary folhcJe. However, atretic secondary follicular thecal cells are

somewhat largcr than th()~e of a healthy ~econdary folhcle, they contain more hpid and

occa~i{)néll autophaglc VélCll()Ie~. The thecal ring surrounding these fol1icles appears

to he mure extcm.ively vélsclliarized, and the con~tituent cells form a dlsrupted layer

(Figure 17). Nuclel in these celIs are generally euchromatlc.

Prccy~tlc and cy:-.tic thecal cells are very sirnllar JO morphology to each other.

The ~IZC ni lhc~c celb i~ slmilar to or larger than the size ot thecal cells of atretle

~ccondary t(Jllic\e~. They arc polygonal in shape. Their cytoplasm contains prirnanly

Iqm.1 dr()plet~, I11ltochondna wlth tubul()ve~lcular cri~tae, and ~ll1o()th ER ci~ternae.

Ali elcctroll dcn ... c halo occa\ltllléllly delineates the outer hOllndary of the hpld droplets

111 thcsc ('l'lb (hgurc~ IHa and h). The nuc1ei ln precystic theral ce Ils are

L'lIl'hromalll', willk Il11CIeI JI1 cystic thecal relis are heterochromatic.

Sccondary Il1tcr~titJaI cells are ohserved m cluster~. Cell size wlthin a single

c1l1~ter IS hlghly varlahle. as i~ cell shape. Generally, however, they are polygonal in

~hapl·. Thcsc cell prolile~ contam an ahllndance ot hpid droplets ot a wIde variety

01 SI/CS. amI I11ltocholldna \Vith tubulovesicular cnstae. NuCIeI are larger and

L'udllomatÎl'. These n~lIs are easily distinglllshed as they occur ln extensively

vasl'ulanzcd c1usters ~urroundeù hy connective tissue relis (Figure 19).

52

Electron Microscopie Analysis of Lipid Droplet Arca

Lipid droplet area was rncasurcd in ten l'clis of cach of the live ccII typl'S

under study, from six dlfferent animaIs. Mean hpld dmplet arl'a + S.E.M. IS

summarized in FIgure 20.

The smallest lipid droplets are those within tllecal l'clis lrom healthy Sl'l"Ollllary

follicles (7.81 + .54nm) and seeondary mterstitial cc Ils (7J·m + 1.(1). Llpid t![Opll'ts

within these cells occupy approximately the ~al11c percentagc 01 thc l'l'II (l1011Il' arl'a

(43.14 + 2.81% in healthy secondary thecal cclls and 45 . .12 + 5.XYi(, 11l ~l'l'Illld;\(y

interstitial cells). Mean liplu droplet area ot prccy~tlc theeal cell!'. is ~\.()2 _t I.~., Il Ill,

hpid droplet area occupying 39.24% + 4.40% of the t()lal l'l'II protrlc 'lIl'a. 'l'hl' IllCall

area of lipld droplet~ withll1 thecal œil:-. 01 atrellc ~t'cond'Ily lollu:k', I~ 1 2.X2 ~ .(JI)

nm, and I~ ~ignillcélntly greater tlwn the Illean Ilplll dropkl ;lre;1 01 hl'i1lthy ilJJd

precystic tolhcuJar thecal ceJJ~ and ~econdary IIltCf' .. llllal l'l'II ... (p < Jl(12). 'l'hl' ~11't' (lI

lipid droplet~ wlthm cy~tlc tlH:cal l'db 1'" !'.Iglllilcantly larger (p < .()5) Ihall IlJo~L' WIIIl1ll

the other tour c la~ses of ceJJ~ ~tll(lJcd, Wlt h a mca n area of J(d)1 ± 2. <)()Illll. II()wl"Vt:I,

the percentagc ot cystlc thecal cell profIle area (hXUpled hy Ilpld droplet\ (51.X() j

8.05%) is not slgnlhcantly dllterent tlmn the pcrct:l1tage ot ccII protlk art::t occuplt:d

by hpid In the other tour cJa~~e~ 01 cell~ examined 111 tht:~e ()vaflt:~.

S3

Arca of Mitochondnal Profiles

The arca of mltoehonùrial prohles WélS measureù in the same cells used in the

analy~i~ of IJplÙ droplet area. Mean mitochondrial area + S.E.M. is summarized in

Figure 21.

Mean area of mitoehonùrial profIles within healthy seconùary follicular thecal

eells is 2.21 ± .32nm, aceountmg for 13.25 ± 1.81 % of the mean ccII profile area.

Mitochondna withm thecal cell~ of atretic secondary follicle~ are significantly smaller

( 1.54 + .1(11111) tl1<1l1 I11ltoehonùna m thecal celb of healthy secondary, precystlc and

cystlc lollJcular oflgll1 (p< .OS). The percent age ot atretle thecaJ cell profile area

()cclIplcd hy mltocllOmlnal proflle:-- (K67 + 1.12%) IS also signiticantly ~maller (p <

.(n) than thl' pcrcentage ot healthy ~econdary, preey~tlc and seconùary interstitial cell

profile alea occupled by l11Jtochondnal prnfJle~. Mean mltochonùnal areas for

precystic (Ut~ + .4~nlll) and cy~tlc thecal cell~ (2.04 + .74nm) are not signihcantly

dJfkrent. Mean 11lItocholldnai area~ expre~~ed a~ a percentage ot the tot, 1 cell profile

arca are abo slImbl (12.96 + 1.1~% and 12.05 + 2.43%, respectively). The mean

are:! 01 mitocholldnal proflle~ wlthin secondary mterstltml cells IS 1.96 + .64nm and

I~ not ~lgflllJcal1tly dllterent fWIll the size of mltochondria withm eells of the four

dassl'~ ot thecal celb examrned. Mean mitochondnal prottle area constltutes 11.24 +

tI.()t)I; of thl' I1ll'an ccII profile area.

54

Analysis of 125I_hCG Binding Sites Using E.M. Radioautography

The specificity of the lahelling of LH/hCG hinding sItes hy 12' l-hC( 1 \Vil!'>

assessed in radioautographs of ovaries expnsed to 1!5I-hCG adlllll1lstl'red togl'thl'I \Vith

a 1,000 fald excess ot unlahelled hormone. In Cllntra~1 tll uvartl'S l.'xposed tll Ihe

labelled hormone only, electron microscopic radloautographs (lI ovafll'S l'XpO~l'd tll

labelled and unlabelled hormone showeù a negliglhk am(ltlnt 01 labelltng (0

grains/l,OOO}.L1l1 surface length, or 1 grain/l O()~m ~urfacc kngt h).

The numher 01 LH hindlng !-.itl'~ per cell \Vas a~sl'~sl'd hy cOlllltlllg tlte Illllllhet

of grains per œil on the Si:lme electron Illlcrmcopic radloautograph!-. lIsed to asse!'!!'>

hpid droplet and mltochondnal area. The nUll1hl'r ot 1251_h(,(; p()~lllv(, !'>Ites ;11('

expressed per IOOOI-Lm surface Jength. The resulfs (mean + S.EM.) arc !'!Ullll1ll1llZcd

in Figure 22.

The mean numhcr ot grains/l000.u on thecal l'l'lb trolll healtlty ~ecolldary (12.1

+ 1.6) and a tretlc ~ec()ndary folhcle:-. (7.7 + 1.)) i~ not 'lgIIIIIC:t nI Iy titi te relit P rL'cy~1 le

thecal cells pmses~ slgntlicantly tewer hindmg ~lte~/1()()OJ.L (.1.7 + .7) a'I WlIlp;lfL:d 10

healthyand atretle ~ec()ndary lolheular thL:cal œlb (p <.(0). m d{) cy~tlc Ihecaln'Ib

(2.0 + .7 reccptors/l000.um, p <.0(1). The mean nlll1lher (lI glillll'" ;1\~()(J;ltl:d wlth

secondary interstitlal celb i!-. 13.3 + 1.4/10()()/-LIl1, and 1\ ~lgIlIlIC;llIlly grL:alcr Illélll lite

nurnher of grains a~s()ciated wlth atretlc, preey~tlc and cy~llc tllecal cel)", (p <.OO(l), hut

is not sigmficantly dJtterent than the numher nI Slte~ a~~()clatcd wlth titeciii cel'" 01

healthy secondary folIicle!'.

55

ElImplantcd AnimaIs - Microcystic Ovarian Condition

Light Microscopie Analysis of Cell Size

A mean index of œil ~Ize of thecal cells from healthy secondary, atretic

secondary and cy~tlc f{)lIicle~, as weil as of secondary interstitial cells, is summarized

111 FIgure 23.

Mean cell slze 01 healthy ~econdary follicular thecal cells + S.E.M. is 9.1 + AJL.

Âtretic secondélry follicular thecal relis élre significantly larger than those of a healthy

secondary lollicle (p < .(01). The me an cell size is 11.4 + .3iJ.. Mean cell size of

Iheca 1 celb ot cystlc ongin I~ ) 1.7 + A}.L, ie. wlthin the same size range as thecal cells

Imm atretlc ~econdary tolhc\es. Secondary interstitial œil slze is signifïcantly larger

than hcalthy and atretic lolhcular thecal œil size (p < .05), with a mean size of 12.2

+ .5J.L.

Electron Microscoplc Identification nf Thecal Cells

The saille three enu'lla med to localize thecal l'l'Ils to either healthy secondary,

atretÏL" ~el'llndary ,lIll! cy!'.tlc lollicb wlthm the macrocy~tic ovary were employed here

wlthlll the I1llCrocystlC ovary. These mclllded: 1) an evaluation ot LM slides bearing

the ~al11l' !'.L'ctioll as the EM gmls; 2) an examination ot the granlliosa cells of the

tolhclc III qllc~tl()n: and 3) the morphological characteristlcs of the thecal œil itself.

Gralllllo~a l'clis wlthin healthy and atretic folhcles in the mlcrocystic ovary are

arc idclltll'al tn tlmse lrom m<lcrocystic ovaries. The membrana granulosa of

mirrocystll' lolheb exhibit charaeteri~tics of granulosa cells l'rom precystic follicles and

56

------ ------ -------------------

macrocysts. It consists of bctween one and four layers of highly irregular. dispcfsl'd

ceUs. NucIei are indented and irregular in shape. and they occupy most of the volume

of the cell. The nuclear indentations, similar to those Ob'iCrvl'd III P' t'CySliC and

macrocystic granulosa cells, are ohserved. Cytoplasll1lc extensions i mliate l'will iht'Sl'

cells, and are not restricted to the apical surfaces as they aIl' in atretie and plL'cySlic

granulosa ceIls. Organelles are scattered throughout tlw ~yt()plasm of tl!l'sC CL'lIs.

Lipid droplets, seen in basal cells of the membrana granulosa ot prccy~.tlc f()l1irk~, arc

not observed here. Autophaglc vacuoles, seen in prc~ystlc gwnulosa l'l'lb, :11 l'

occasionally observed in these eells as weil. Junctiorml c()ll1pll'xe~ ail' r'lrcly

ellcountered (Figure 24).

Electron Microscopic Description of Thecal/Scc'HH.laI.Y..-lntcrstitléll ('l'II

Morphology

The morphology of thecal and secondary mtastiti,ll œlls in microcystll' ovancs

does not appear to be different from the structure 01 anal()gou~ n'lb wHIlIn

macrocystic avaries lIpon inspection. Ultrastructural (lJffercl1ce~ hetween œlb wlthlll

the two ditferent types of cystic ovaries becomes apparcllt only after qu:ml,t;ttivc

analysis of lipid droplcts and mitochondnal areas was complete. The~e dJl k/l~llœ~ WIll

be discussed. Electron microscopie radioaut()graphs 01 él nllCrocy!-.tic thecal ceil (Figure

25) and a ~econdary interstitial cell l'rom an ETimplantetl oV(lry (Flgure 2(») are

included for comparison.

57

Electron Microscopie Analysis of Lipid Droplet Area

I,ipld droplet élrca was measured in ten eells of each ce]) type under study,

from six difterent animais. Mean lipid droplet area + S.E.M. is summarized in Figure

27.

The mean an~a + S.E.M. of lipid droplets within healthy and atretic secondary

foilicular thecal ccll~ :/nd ~ec()ndary inter~tltial relis is 8.33 + .57nm, 9.82 + 1.04, and

9.68 + I.04nm, respectively. There are no ~lgnIficant difterences between these

vélllle~. llowever, the percentage~ 01 cell prolile area occupied hy lipid droplets Ifl

healthy sewndary theral cells (52.61 + 2.75%) and ~econdary II1ter~titial cells (55.64

+ 3.46%) are ~igniltcantly larger (p < .(3) as compared to atretic secondary theeal

l'db (41.15 + 3.40c;~,). The l11ean area of hpid drop\et~ in cy..,tlc thecal cells IS 16.46

+ 9811111, élnd I~ ~lgnIlicantly larger than for healthy ~econdary follicular thecal cells

(p < ()'()()6), atrL'tl<: ~ec()ndary foilicular theca (p < 0.0(7) and secondary interstitial

l'db (p < O.O()Cl). The propOltJon of Cy~tlC Illecal cell protJle aren occupied by hpid

({lJ.50 + 4.2(lr) I~ ~lgl1lrlcantly greater eompared tn healthy and atretic secondary

foiliculu theral cdb (p < .()~).

Arca (JI Mltocholldrwl ProlIle~

Arca 01 Illltorhol1dnal prohles was l11ea~ured in the same eells used in the

an"ly~l~ 01 IIpld droplet area. Mean profile area + S.E.M. for each of the four cell

typl'~ L'\al11l1lL'd IS slIl11marized in Figure 28.

58

Mean area of profiles within healthy secondary follicular thecal l'l'Ils IS \.l)l) .±

.16nm, or 11.15 + 1.22% of the mean ct'Il prolile area. Mltochondnal plllflll's wltl\ll1

thecal cells of atretlc sccondary folliclcs average 1.71 + .15nl11 III alea, or 9.7h J'.

1.34% of the mean profile area of these cc Ils. Cystll' nlltochllndnal illca I~ I.X4 +

.10nm, whlch corresponds to K27 + \.17% 01 the meall total pl nille arl'a, l'hl' 1llt'.111

area of rnltochondrial profiles withll1 secondary mterstltlill l'cil I!\ 2.2h .t .2·111111, ami

is significantly larger than mJtochondnal proilles of atretlc and cY'" Il' Illllu:ulai llnglll

(p <.(4). The large standarù l'rror lor thl~ œil populatl()11 rclkrt!\ tlll' gll'atl'I

variahility hetwecn the!\c cdb a~ compared ln Ihe olher l'l'II type". 'l'hl' IWlet'lItagc

of the seconùary II1ler!\tltlal cell pro/rie area occl/pled hy IIlI!ocholld":1 " 10.-1/) !

1.64%, Therl' a re no ~Ignr / J(:ant d Itterel1Ce~ hetwec 11 the pr(lplll t Il)Jl l" 1 hl' lolill l'l,II

profile area occuplcd hy milochondrial pro/J1c~ III the~c IOUI l'l'II type .. Wllhlll il

rnicrocystic nvary.

Analysis of /251-hCG BlIlding Site .. U~lI1g E.M. Radloillllog[i.Wll)'

The numher of LH/hCG hmùlI1g ~Ite~ per œil WiI!\ a ..... e ... ~ed III the ~al1ll' manlll"1

as described tor macrocy!-.tic ()vane~, and I~ expre~ ... cd per 1 ()()()/-L1ll ~lIr1ilCl' ICl1glh. 'l'hl'

results (rnean + S.E.M.) are ~ulllmanœd 1/1 Flgurc 29.

Healthy ~econdary tollJclllar thecal ccll~ have an avemge ()I Kit 1 1

grains/l O()()/-L/ll. An average ot (d, + 1.0 gnlll1!-./100()/-L11l .Ire as!\oclated wlth atletH'

secondary tolllclllar thl'cal cel"". Cy~tlc thecal œlb P()"'~c",!-' an i1Vl:filge Cli .4 ± .1

LH/hCG sltes!1000/-L1l1, which I~ ~lgnilJcantly Ic~~ thélll the number of ~Ite!\ on the tlnec

59

othcr ccII tYre~ examined in these ovaries (p < .001). Secondary interstitial cells have

a ~JgnÎfÎeantJy Jargcr numhcr (16.J + 2.4/10()OJ,Lm) of LH!hCG receptors as compared

10 atrctÎc ~ccomlary and cystJC thtcal œlls (p <.0(4), hut not as compared to healthy

~cc()nùary theeaJ cclJ~. ThÎ~ may he ùue to the hlgh standard error associated with

grain count., for ~cc()ndary intcrstltia! eells.

60

Macrocystic vs. Microcystic Thecal and Secondary Interstitial Cells

A comparison of cell size, Iipld droplet art'a. art'a 01 111Itochondnal plOlill's .\Ill!

the number of 125I_hCG hmding sltes/lOOOp.m ~urlact' kngth hl't"'l'L'11 l'L'Ils wlthlll

macrocystic and microcystlc ovanes wa~ made III mLier to ellll'ldatc l'l'lIl1lal lhlll'relll'l'~

in these two ditterent P()lyCy:~tlC ovarian condltiolls. Thl'~l' fmu palallll'tl'I~ Wl'It'

assessed indiviùually hy ~tatl~tlcal1y comparing measurcllll'nb ohtallll't1 lOI healthy

follicular the cal cells. In tho~e m~tancc~ wherc the paml11l'tn UIllIeI ~tlltly w.t~ 11l1ll1d

to he the same ln healthy secondary thecal cell~ lrom hoth man ()cy,tll' and illiCH H'y,tll'

ovaries, atretic thecal ce Il. cy~tle thcl'al ccII and ~ccondary Illtcr'tltlal l'l'II vallJl'~ lin

the parameter were comparco. Thc only parameter lor whlch a ~lglldlC.tllt thllL-lTlIlT

was found hetween ht'althy secondary lolhl'ular t11ecal l'l'II, (lI maCJ(Jl'y!\tH: ill1d

microcy~tlc ovanan origm I~ the llulllhcr 01 1.?51_hCG hllldlllg ~ltl'~/1 ()()()p.1Il !\lIllilt't'

length, whlch was IOllnd tn be slgllIlll'antly larger III hc;dthy !'>l'COI1t1.lIy thl'tal l't'lb

WIthin EV-treated ovane~ (p < '()4). Thl!\ may retlect dlllclCIltT!\ III the 'IK'nllt'

activity of the ligand. ft i~ lllore hke\y udC 10 the laet tllat the endogclHlll!\ LI 1 Icvt'b

in microcystic PCO are hlgher than m mélcfocystic PCO (MeCII tlly et al, 1 (]X(),

Gro~ser et al., 1<J~7; McCarthy ami Brawer, !tJX<J), wlllle the ':lJl1l' <IlJlOllllt (lI Ilg:lIJd

was admini~tered In to dl11mab 01 hoth mode\~.

Cell ~ize. lipid droplet area and lllltochondnai prolIle area are !\lllltlar III ht.:;dthy

secondary thecal cell~ 01 macrocy~tlc and mlcrocy~tlc ovanan onglf1 (p > J)())

Becallse atretic lolllc\ _~. cystlc t()lIicle~ and ~ec()ndary lllter~litiai celb aIl ame lroll1 the

61

- --- -------

dlffcrent developmental pathway~ ot a healthy secondary foilicle (Erickson et al., 1985;

Brawcr ct al., 1 <JR6), thc!'lc thrce parameter!'l can he compared in arder to elucidate

flCltcntial dlffcrcnces bctween macrocystic and mlcrocystlc ovaries.

Analyw, of CcII Slze

Atretlc ~ec()ndary thecal cell!'. in macrocy!'ltic OVélnes (12.20 + .3,um) are

slgniflcantly larger (p < .0)) than those In microcy!'ltlc ovaries (11.4 + .3,um).

Seconuary I/ller!'ltltJal œil!'. wlthm microcY!'ltic ()vane~ (12.2 + .),um) are slgntficantly

larger (p < (3) thall the .,ame celb lrom macrocylltic ovane~ (11.3 + .3,um).

1 -'1ml Dropkt Âre;!

Thcl c arc no ~lgl1ltJcant dI!lercnce!'l hetwcen the mean area ot lipid droplet

prollle!'> or the percentage 01 mean œil profile area occuplcd hy lipid in cells from

Illarmcy!'>tlc ovanes compared 10 thelr c()unterpart~ 111 microcystlc ovanes.

Arca 01 Mltochol1dnal Profiles

The mean area of Illltochondriai proflle~ in macrocystic thecal celb (2.04 +

.74n111) I!'I slgJ1lfïctlntly larger than the mean profile area In microcystic thecal cells

(LX..f + . IOn III ). The perœntage ot the œil profIle area Dccl/pied hy nlltochondrial

p/llflll'!'> I~ abo !'>lgJ1lllrantly largcr 111 l11acrocy~tic theral cciI!'. (12.05 + 2.43% versus

K27 + 1.17( (). Dltkrence~ I/l Illc(\n mitochondnal proille area and In the percentage

01 ~l'mlldary JI1ter~tlllal ccII plOflle area occl/pled hy I1lltochondria are not significant.

62

DISCUSSION

Alkaline Phosphatase EXprl'!'>!'>IOI1

Alkaltne phosphata~e l!'> an e1l7yme 1I11pmtant 111 rl'actlons ll1\'Olvlllg thl' syllthl'Sl~

of steroid hormones (St:lIford et aL, 1947). A!'> wd!. it l!'> heltl'\'l'd to hl' nl'n'"aIY 101

the passage ot ~terolds acro~~ ccII I11l'l1lhral1l'~, a!'> tlm, prorl'~~ rl'qulll" pIHl~phatL'

esterificatlOn (Moog, 1(45). In the rat, al ka 1 III l' phmphat"'l' pmltlVl' rl'll, Illrludl'

follicular thel'al Cl'1I~, intertoiltcllidr !'>trom:ll l'clb, 'l'wnd:IlY mtl'( 'tltlal l'dl, ami

vascular endothellal cell~ (Demp,ey et al., l').~(); Klllggl' and Il'athcl11, 1 (»)·l; MrKay

et al., 1<)(,1; Jacohy, 1962: Kent (1 III 1 Ryle. 1(>75). (i(anllima l'l'II, arc dl'VOld 01

alkaltne phO\phaw,e activlty (Knlgge and Leathclll. Il)54; Kent ami Ryle. 1lJ75).

The e\rre~~l()n of alkallne phmph:lta!'>e appeélr!'> tn Il'Ilect t(()PIC hOlllHII1l'

~timl1lation ami !'>teroldogene~l~ III tl1ecal Cl'Ib ami tlll'cal dellvdtlvl'~ (MrKay ct aL,

]<)61; Pmkerton et aL, 1%1; Kent, 1974, R(l~s, 1(>74; Kent and Ryle, 1975).

AdmllllstratHlll ot L11 and hCG tllrther enll:lllCe~ alkillJnl' phmphata~c élCtlvlty III l'L'lb

which are aikalllle pho~phata~e pmltlve (Kent ilnd Ryle, 1(75). The adlllllll\tléltlOIl

ot powdered whole pltllltary to hypophy~l'ct()llIl/l'd ,lIlllllab I~ lollOWl'd hy

enhancement or re~toratlon 01 alkalllle plw\pllata ... e al'tlvlty tu dl'veloplllg tol"l'k~ ;1Ill!

newly tormed corpora lutea, whlle corpora lute:! III tlte ovary Whlcll Wl'rC tormel!

hefore hypophy ... eclomy ùo not [(.:c()ver aikallllc phmphata ... e actlvlty (Ikmp\l'y ct aL,

1949). Hence. the alkaline phŒphata~e actlvlty ot a œil may abo n.:tlect It\ ~tate of

differentlélt Ion. ln human OVélfle~, Dlmm and Mende (I<JS(I) lound alk:drnc

rhmphata!'.e actlvity ln ~cc()nùélry interstltial celb to he inver~ely related tn the lipid

content of the cel!!'., ~lIgge!'.ting a rm~ible correlation hetween alkaline ph()srhata~e

actlvity amI IlLJctllatl()n~ ln hormone prmlllction. Thll~, all<alme rh()sphata~e is a

valuahlc to()1 lor (l',~e~~lI1g troplC hormone ~tll1ll1latlon anù ~teroiùogenic activity in

thecal cells and thecal deflvatlve~ ln p()lycy~tlc ovarie ....

ln the pre!'.enl stuùy illkalme phmphala~e ilCtlvlty, locahzeù III control,

Jl1acrocy~tlc ;1IIl! f11/CroCy!-.tIC ovafle~ u~lI1g an 111111l1ln()hi~t()chelllical marker, IS oh!'.erveù

III thecal cd"- (lI he;lIthy and atretlc pnnwry and "l'condary tollH.1c ... , in corpora lutea

(Hl control (IV.!! le!> only), and III \'a~cular endothellal l'l'lb. The relatIve e>.pres~lOn of

alkalllll' ph()!>phata~e in the~e !>trllcture~ , ... ~lInJiar to that rl'porteL! 10 the hterature

(Del11p~cy ct i11, 1<J4lJ, Kl1Igge allL! Leathelll, IlJ5-l; l\.1cKayet aL, 1961; and Kent anL!

Ryle, 1'>75). The thecal wreath of l1lacrocy~tlc and I1llm)l'y~tic tolhcJe~, m contrast

10 ellher pnmary or alllral, hl'iIlthy or alrdlc lollJcle~ and va!'.cular enùolhehal l'clis,

~lll)w!> Vl'Iy 11111e alkahne phmphala~e é1CIIVlty and that which IS pre~ent IS pfllllanly

locahzcd ovcr capJllary enùolhchal cclb. ThiS ~lIgge~b that lllacrocy~tJC anù

!11lcrocy~lll' lollJ('lc~ arc not stclOldogenrcally active. Furthermore, the decrease in

alkalll1c phmphilla~e e>.pre~~iol1 III theCllI cells of cy~tlc tolllcles, as compareù ln that

111 allelie ~l.·l'(Hldary lollIcles, Illay IIldH:ale that tbecal celb in the~e two structures

lollmv dlfkrent patl1way~ of drlll'rentwtH)Jl, Ieadlllg to drllerent tunctlOnal capacHles.

1IIII1l1ll1oslarning ot ~ec()l1llary ,,1tcr~lltlal relis for alkahne pllO~phatél~e I~ hlghly

vanahlc hetweell control, EV -trl'aled and E:-Illlplanted ovmies. Alkalrne phosphata~e

positive interstltl<ll cell c1usters in macrocystlc OV<lm's are largcr. more nlll11CIllll~, and

more inten!>e1y !>tained than thme 111 control ovant'!>. l'hw.. !>t'l'ondary IIller!>tltl:!1 "clls

in macrocy!>tlc o\'ane!> may he elther 1111Hl' !>L'mltlYC III tlOpll' hlllllHlIlL" ,11I1l1l1.ltIl1ll.

more activc III !>ll'roldogenc!>l!> thall thelr cOlllltcrpart 111 a l'yl.'llIIg O\'.Ily, 01 bulh.

Immunostall1lllg 01 ~ec()ndary Illll'l!>tJtl;t1 l'l'lb III IllICloCy'tll' oyallt'!> Ill! alk.lIlIH'

phosphata~c 1" lucallzed tu !>mall. IIllcml'ly ~Iallled 'tralld ... 01 IIlICI~lltlal l'l'lb a III 1 hl

small. round. hir.hly rl'actlve ccII c1ll',Il'r!>. Ill'lther 01 whlch ale llh~l'IVl'd III l'lllltllli 01

macrocy~llc o\'ane". These Il1ler~lltlal cclh mav pm"'lhly hl' dCII\'l'd 1111111 a

spontaneoll"ly Illtl'll1lzl'd lolllclc. or from ~plll1t;llll'llll~ly Illll·llll/l~d. hlghly ... tl'lllldllgl'lIIl.'

secondary II1ter!>tltlal cell clll~ll'r". A~ seClllldary II1tt"!"!>tl!lal l'l'II ... 11I11111.lIly ;1I1\e trulli

the thecal l'l'II layer 01 c()lIap~cd atrellc lolllck~ (revlcwl'd III biChOIl. 19X5), il III 1 a ...

control and lllal'rocy~llc ovane ... lh, Ilot cOlltalll "l'COllllttry IIlICI!>tlllal l'Cil lhl .. ll·l~ lhal

rl'semhle thml' 111 the IlllcroCy!>tlC ovary 111 l'lthel morphlll()gy or alk;lIl1lc phll\phdla~c

expre~~IOI1, Il I~ po~~ihle thal thecal ccII preclIr ... or~ m;IY lolillw éI dillerent palhway lit

cytoditferentliltloll in Ihe~e E,?-treated ovanes, glvlllg me llirectly 10 olllllgl'lIlcally

distinct, slenmlogenically active ~econdary II1lel ... tlllal cell~.

Cell SI7e illld LI I-BlI1dll1g CilpilClty

Thec;I1 celb and secomlary II1ler~tllliti cclb ;Ire dl.,!>lt Icd l1lorphologlcally a~

either normal ()f hypertruphled. 1 Iypertrophlt'd cell~ ;Irt gtnerally c()n~ldertd to hl'

highly ~teroid()gtIllC (Carither~ and Gretn, 1 (J72i1,h; I~nck~()n, 1 ()X5). 1 Iyptrtrophy I~

one of Ihe earlle~1 chélnge~ ln Ihtcal ccII l11orphology il" atrt\\a htglll\ (LJJitnl>rock et

al., 1lJXO). A!'> é1trcc.,ia a dV<HlCC !'>, a dcerea!'!c 111 Ihe nllmher ot lropie hormone reeeptors

occurs (U IIcnhrock ct al., 19XO). RIchard!'> and iJiJdgley (1976) propo!'>e that tolhelllar

atre!.léI I!. cilll!.ed hy fi decrea!.c ln ~cn!.ltlvlty ot theca\ eells 10 gonadotruplc hormones

(Rlchard~ :tnd Mldgky, Inn). Alter an alretle tnllicle C()lIap~e~ and II~ thecal cells

arc e!'!lahllc.,hL:d ;I~ ~ccondary IJ1ler~tltl;t1 cell~, the numher of tropie hormone receplors

IIILrca!'!e~ é1galll, ~llrpa!.~Jng Ihe Humber po~~e~:-.ed hy alrellc fol\lcular Ihecal cells

(Shaha and (;rccllwald, 19X2).

ThL' cap;lhJllty (lI a œil ICl hllld L11 I~ e()n~idereù ln rctleet tmple hormone

~L'I1~lIlvlty ilnd III hl' an Index of the c.,tCroll!ogcIlIC cap;IClty ot thecal élnd ~econùary

I\1Il'r~tJtlal l'l'II" 111 the ovary (Dllmnn. 1977: DIl11lnn and Ikrman. 1lJ7lJ; Rowe et al..

ILJXI and Il)}{h) Cla~:-.icillly. ccII :-Ize and LII-hIlH.lJng capaclty are con~\{.lereù 10 he

vaillahk tOlll~ lm a~~e:-':-'lIlg the !.tcrold hlmynthetle actlvlty ot theeal and ~econùary

IIlll'rstitlal l'l'lb Paradoxlcally, thecal hypcrtrophy and ~econdary Il1terstltléll œil

fm matl()l1 III hYPIlI'hy!.cctol1llzL'd rat~ are aCl'Olllpallled hy an increase ln the 1111rnher

Clf 1./1 rt.'l'l')lI()1 " imd an IIlCfl'a:-.e 111 !.lenllllogl'l1Ic aCllvlty (Hillier and Ro!.s, 1979;

EIICboll. IlJX 1). In the prescnt !'ttut/y, œil :-.ize and LI 1 hmdlllg are evaluated in thecal

,IIIL! ~l'l'orlllary inlerstltlal relb Hl l11acf()cy~tlc and 111lcrocy~tic ovaries, A cell's size and

II~ 1.1 1 bmdlllg capal'lty WIll he evalllated in col1JlIl1l'tion wlth alkalme phosphatase

l."plL'~~IOIl :1" ail tndex ot Impie hm mone ~l'nsitlvJty and putative ~terolùngel11c

capal'lly. ('cil ~llC and Lli hilldlllg characteristlcs alone WIll not he regarded as

ah~()llItc IllllJl'alllHl~ ot a ccll's !.tcroid biosynthetlC activlty.

66

In the 1l1:\(:rocy~tic ovary. the ~ize ot atretlc. prt'cy,llc ilnd cystic thl'l'al n'Ils ilnd

secondary inter~tltial celb I~ approxlIllatdy thl' ~al11t', IlmVt'wr the 1l1lll1hl'r III 1.11

receptors per 1.()()()J.L1l1 ~llrfal'e Iength. a~sc,st'd u~lIlg radlOillitography \Vllh I:\I-h('(j

as the ligand. i~ not the ~al11l'. Secolldary 1I11l'r~tltlal cell~ rOlltam ~lgl1lltr.lIltly ll11lrl'

LH-hlllding ~Ite~ than any ut the Dlhl'r l'l'II, l'x.tl11l1ll'd m tlll' llV:lIy. wlllle pll'l'y~lIr ilml

cystic thecal l'db wntain ~lgl1llll'ilntly kwer hllldlllg \ltl" than healthy ur atll'tIC

follicular thel'al l'ClIs and secondary inter~tlliai l'elb. Thll~. l'l'II ~1J'e III the~l' OVillll'~

doe~ not a ppea r tu rl'nect tmplc hormune ~e n~lt IVI ty ur pu ta t IVl' ~lerOld()gl' Ille ::rt IVlty.

A~ precy~tll and cy~tlC tolhl'lliar theral l'db pm~l":-' Vl'ry kw 1 JI bmdlllg ~llc,. olle

wl)Uld predlct tlwt they me not Lli M~Il~ltlve ami thl'Il'tole arc not IIkcly actlVl'ly

synthe~lz1l1g androgen~. The 111 III 1 III a 1 al11lHlIlt~ ot" alkalll1c phmphat(\~e III thc:-.e n~lb

al~o sugge~t~ thl~. C()nver~ely, thc large ~l/e ot the celb, thL' V(\~t nlllllher 01 1 JI

hmdmg ~Ites. and the pre~ellce ot alkallTle ph()sphata~e 111 ~e('()ndary II1ter~tltlal rcll~

in macrocy~llc ()vane~ ~ugge~t~ Ihat Ihese l'db arc potelllially very active III androgen

synthesi~.

In microcy~tic ovarie~, ~econdary IIlter~tllllll ccll~ arc ~ignilll'antly larger than

healthy, atrellc and cy~lic loiliclllar thecal cl'Ils. They also pm~ess slglllltcantly more

LH hinding ~ite~. Thll~, microcy~llc ~ec()ndary 1I1ler ... tlllal ccll<. appear 10 I)l' 11Ighly

~ensitlve to lropie hormone ~tlJlll1lat((JI1 and thll~ potenllally very al'llvely ~tef(lIll()gelllc.

Alkahne phmphala~e expre~~I()n hy the ... e celb "Llgge~l!'. the ~aml'. De:-,plte the

simllarity III the ~ize ot aIrelle and nll(:rocy~lle Ilwcal celb, the latter conta1l1

slgmticantly kwcr LH reccptor ~Ite~, parallekd hy an apprcciahly smaller amount of

alkalInc ph()\ph;lta~c. Ba~ed on thc~c llh!.crvations, It appears that these

hypcrtrop/lIl~d cy~IIC tl1ec:al cell:. are functlonally di~tJnct from atretic thecal cells, heing

le!>~ !>CIl\ltlvC to tmplc hormone ~tmllJlatlon and !.eelTIlngly le!>s androgenic. Perhaps

tlley reprc\l'Ilt ;t tcrmlllai !>tate of alre:--Ia

Mltocl1(Jfl(lnal and Lipld Droplet Proille Ar~a!'>

MJtocholHlna provlde a unique method lOT lTIol1ltoring !>ome of the temporal

etlcct~ ot gOJ1i1l!otropIC Ill)rl11()ne~ on ovan:m ~terOldogene~I~, al. weil as on the state

(lf dlllerclltlatl(Hl <lf lolllcular luteal tl~~ue. Lli ~tllnlllat\(m 01 luteal and ~econdary

IIlterstltllll cel'" lrom nornwlly cycIlng ovanc:-- re!>ult ln mitochondrial changes.

Strllcturally, Illltochol1ùn:I hecome more wlinded 111 !>/lllpe, and the cmtae change

lrom a lamellar to a tuhulove~J(:lIlar organlzation (Dil11Jno et aL, ] 979; Rowe et al.,

I<JH 1). Thl'~l' l.tructur:tl change~ arc accompalllcd hy fUllctlonal changes whlch IIlvolve

the productloll 01 ~peCJtïc protein~ lI11pmtant lor slt:roid()gene~is, inclllding a

choll'~tl'rol ~Idl' dWIIl c1eavage complex whlch catalyse!> the rate limlting ~tep ln

stewldl)genl'~'d:-- (Omura ct aL. 1 96(): Rowe et al., 1 <JX6). ThiS mcrea:--e in protein

~ynth~sis I~ act'ompanicd hy an increased ahllity of mitochondnèl to convert

prcgl1l'IHllollL' to prog~~tcr()nc (DII1l1nO et al.. 1979; 011111110 and Berman, 1979; Rowe

ct aL. 19X 1 :lJlll 1 ():-;()), Mllocho!ldnai sterOldogenic actlvlty IIlcreases with follicular

dl'wlopl11l'llt (DlIllllHl. 1977: Rm\e et aL, ]<ml), a pr()ce~~ whlch i~ parallekd hy

hllther cyt(ldlfklclltlatio!l of theral cells (revicwed in Ericbon. 1(85).

6X

Removal of LH stlmulatJ(m of the ovary hy hypllphysl'ctlll11y rl'sults ln a

reduction in ~Ize, numher of mitochnndna and the numbl'r ni l'mtal' lf1 mitlll'holldlla.

Furthermore. 11lItol'hondriai l'mtae are re~trlll'tllred t'rom a tlibuloVl'sirular tll a

lamellar orgal1lzation dunng the IIr~t l1lonth followltlg hypophy~l'l'tomy (Canthl'Is allll

Green, 1972(1). A~ weil. the amount 01 Iq)1(.1 l'olltallll'd wlthlll ~l'l'ol1d:lIy Intl'I~tlllai

cells increase~ appreclably following hypophy~l'rl()llly (C'alllhl'IS and (Jll'CIl. 1'J72a,h).

As mitochondna and Itpld droplet~ are holh II1volved 111 .,Ieroll.! ~ynlhe~ls, and a~ Ihl'

size ot hoth I~ alleeted hy LH, mitoehonùnal and Iqml ll!llpll't area aie a~M'~"' .. 'd 111

the pre~ent ~llIdy tn provide an aù<.lIllonal index 01 llOpll' hormolle .,llIlllllalloll and

steroidogelllc l'apacity 01 thee:!1 and .,el'<lndary 1IlIer~llll;d l'l'II., wllhlll poly("y~lll' ovallcs.

Healthy. atrellc, precy~llc and l'y~tlc lulllculal thec:!1 cells and Sl'U>lHlilly

mterstitial l'ells wlthm l11al'rol'y~tic ovanes ail ClJl1talll nlllllChlJndna wlth tllhlllovnlclIlar

cristae. The area 01 mltochondnal prollle ... 111 <ltlellC loll1l'ular thecal l'l'Ils 1:,

slgnificantly ~l1laller thelll Illltochonùna wlthlll thecal cell~ 01 healthy, pll:'ey~tlc and

cystic llnglll. The prop()rtlon 01 the l'l'II proille area occlIpled hy 1ll11()ch(lIIdllal

profiles i~ ~lgl1lll\:alltly ~maller in the~e celb a~ weil, .,lIgge .... lllg Ihat they alw P()~~l'~~

tewer I1lltochondn<l than healthy theeal, precy~tlc theeal, cy:-.tlc theeal and ~ee()lIllary

interstltJ(\1 Cl:'11~. The~e atretle loll1eular theeal œil., abo cont:llll larger IrPIt! dr()pkt~

than healthy and rreLy~tlL thecal œlh. B;I.,ed (lll the rep(J1 t., (JI CanthcI'I ilnd (irl'clI

(1972a,h), the Ilumher and ~17t: 01 11lItoc!Jollùnil anù the ~ll.e (JI I1pld dropleh III the~c

atretic tolheul:tr thecal cell~ do n()t lIt the typleal prof Ile 01 éI L11-.,tllllulated,

androgenie ovarian œil, a!o. the numher of LH binding sites and alkaline phosphatase

expre~~)(Jn ~llggc~t that they élrc sensitive tn tmpie hormone stlmldation and élre

androgenic. Mitoehondnal and lipid droplet mdices m these ce Ils, relative to other

thecal cell ... 111 thls ovary, ~lIgge~t howevcr tllat the~e celb cOllld he partially deactivated

ie. on the p;lth tn lI1activity, whlle cy~tlc thecal celb may represent the cornpletion of

the proce~~ ..

Macfl)cy~tlc lolllcles, ln rnarked contra~t tn atretic thecal cells in EV-treated

ovancs, pm,~~~~ n~gllglhle ampi.lIlts ot alkaline phosphatase activity and very few LH

hindmg ~It~~. They abo contain the large~t hpid droplcts. These indices together

support the alblllll' rho~phata~e data Illdlcatmg that m{\(:rocy~tic thecal cell~ are

sten)J(log~llIl':dly IlIactlve. FlIrtherl11OJc, they mdlcate 1hat macrocystlc thecal cell~

arc tUIlCtlOIl:Jlly ,llId 1Iltrastrllcturally dl~tmct trom atrdlc tolhcular tbecal celb.

ln I11ILT(Jl'y~tll' ovane~, the large~t l11itochol1l1na are round m ~econdary

IIlterstitlal l'dl~. IloweVt'r, the proportioll nt the total cell protile élfea occupled hy

I11ltocholllh 1;11 plOt des IS relatlvely con~tant betwecn Ihe four ccII typc~ exammed.

Nonet hek~\, ili ":lIl/1e phmpha la:-.e e:\prc~!->lon, the capacity tu hmd LH, cell Slze,

Illllochondilai ~li'e and ultra!->trllctural orgaI1lzatioll ;111 ~lIgge~t that these cells are

~yl1thetlcally acll\'L' and thal they prohahly cOlltnhute ~lIh~tantially to the plasma

~tt'rOid hormonl' pmtde Jl1 thl'~t.: animais.

Cy~tll' thl'l':llcL'lb nmtaill ~ignIlic:llltly larger IIpid droplets than healthy thecal,

:lllelic tliccl!. :tlld ~L'L'olldary mterstltial ce Ils. In additIon, a slgnificantly larger

70

proportion of the œil profile area in microcystic theral l'l'Ils is lll'l'upicd hy lipu.l.

Alkaline phosphatélse activlty and LH-sensltivlty arc minllllai 111 tht'SC Ct'II~. Tht'~l'

observations, together with the ahunùance 01 lipid. sllggt'~t that as was the l"a~l' fOI

macrocystic theeal relis, mlcrocystic thecal l'dIs are \lOÎ stclOIdogemr. Tht.' luhuÎ.1I

organiza tio\l of 111 itochondrial cristal' in tlle~e cc Ils, m in l11al"ml'y~t le t lwl'ili ce Ils, aga III

suggests that they are not terminally dcactlvatcd.

How are l'ysts relélteù to nOler lollicular structurcs'!

The morpholob'Y of the follicular ~trllctllre~ 111 EV-trcatl'd and E!-II11planted

polycystic ovaries show signs of atre~la. The cha nges 111 f olllclliar dynamles plCl'l'dll1g

the development nt the pnlYLystic morphology sllgge~t that cy~t formatIon IS a

consequence of an alteratiol1 or <IlTcst 01 the atretlc pr()ce~s (Br "Wl'I ct aL, l 'JHh,

1989). The morpho!ogy ot prccy~tic lollJclcs, a~ weil a~ the L11-hll1dlllg cap:lClly :tIld

degree of alka!ine phŒphatase exprc~~illl1, which arc Illtefl1lt'dlale hetwet'n atrelle and

cystic folhcular characteri~tics, support this. As weil, the ll1emhrana gfallul()~a (Jf

precystic tolhclcs IS characteristlc ot a lolhcle 111 .111 advanced ~tate of atre~la (Zl'Ikr,

19R4), conwlIllllg lipid droplets lf1 the penlllurai layef, exhlhltlllg il dl\fUplu)f) of

junctional complexes and showlIlg cytopl(l~I11IC hlehhll1g (lf the apIcal Sllfface~ ()f the

periantral layer of cell~. The melllhrana gr(lnul(J~a (lI d lllacrocy\t 1~ dl~t1l1ct 1 rom th:lt

of an atretic or prec)'''itic tolliclt.~ and, altllOugh the cell~ are Irregular and not

functional, they do appear to he ~tahle.

The tran~lti()n trom the precy~tlc tn the cy~tic condlti()n cOll1cides with il Ims

7]

of hCG h/lldJl1g ~Ite~ and, pre~umably, a loss in sensitlvi~y tn LH. ThIs Joss of LH

hlfH.Jing ~Ite~ IJkcl) re~ult.., in a dlJllJl1ution in androgen production. The lack of

,llkahllC phmpll(lta~e activIty and the ahundancc of lipld In these cells lends support

10 lhl~ Idea A.., cxce!'l~lve local concentrations of androgen appear to he instrumental

JO Illarklllg ... pecIllc f(lllIde!'> for atre!'lJa (Louvet ct al., 1975), an attenuatIOn in local

androgen c()nl'("lltr.ltlon~ in the~e follIcll~!'> may re!'>ult JO ail arre!'lt uf the atretic process

and the ,tahJlllcltHm 01 the altectcd folhclc a~ a relatively inert cyst which, because ot

Il!'> large !'>I/l\ 1\ unahle 10 Cllllap~e.

Mlc!()cy~tlc t!wcal lelb abo ~how mimmal alkaline phosphatase élctivity and a

kw LlI hllldll1g !'>lIe~, a!'> do macrocy~tic theca! relis. The memhrana granulosa of a

111 icrocy:.. t le loi Il dl' IS not Ident Ica! 10 t ha t of a m:lcrocyst, but rather pO!lsesses

charaetcl i~tll'~ (lf the lllel1lbran:l granulo~a of both precy!'>tll' and macrocystIc follicles.

f)lllelenCl'~ hl'lwl'cn the morpho!ogy of microcy!'>til' and macf()cy~til' f(}lIicle~ det:llled

III thi:-. paper, a~ weil a~ the ~ize of the mlcrocy~tlc follIcle [(me the question as to

whether ()] Ilot Il IS pO~~lbll' that the Il1ll'rocy~tic tolliele I!'> merely an atretIl' follic1e

]11101 to c()lIap:-.l'. Furtl1er studies me required 10 dClerlllll1L: tlw,. A~ ~ome seconùary

IIlteNltlal ccii CILJ\tl'l~ exhlhlt the approxlIllate ~hape and ~izL: 01 mlCrocy!ltll' follicles,

ami as IJlllÏnry~tlc tollll'le!'> ~1I11 hL'ar a re~el1lhlallCe l() atrellc follicJes, it i~ possible that

l1lJl"rory~tlc Inllick!'> arc mere!y degeneratmg 1()IIIc1e~ w!lIch tollow a difterent atretie

pathway. hut tn the ~al11e end. le. tn hL:col11L' a ~econdé\ry interstitJaI ccII e1uster.

Sl'clllHlary II1ter~tltJaI celb are lar more nlll11eroll~ III lhe~e E.?-implanted ovanes, as

72

compaIl:d tn EV-trl:ated ovaries. The collapse of Il1Il'rOCYSts and the pl'rSish.'Ill'l' of

macrocysts may account for this in part.

On the ha"l~ 01 morphology, the Illllllht'r of 1.11 lel'eptors, and the nUlllhl'1 of

ce Ils which contain large qualltltles 01 alkallIIe phml'hatasL', Ollt' would prl'dll't that

the polycystlc ()\'ary ln the E~-lll1plalltl'd rat I~ lai more iIl·tlve III synthl'sl/lIlg

androgens than I~ it~ counterpart III the EV-treated 1ll0l1e!. Il local and l'irculatlllg

androgen kwb are 1!1 tact tllgher 111 E:-Il11plilnted rat~ wlth pen, thl~ wOlild havL'

Important COIl\l'queI1cl's on pltLlltary IUIlCtloll amI on thl' n.prl'~~i()n 01 ~l'Wllll.lIy ~L'X

characteristll's in the~e rats. TllI~, however. rel11alll~ tu hl' dctl'rl11l1lcd.

73

REFERENCES

Adam:-., J., D.W. Pobon, N. Ahdulwahld, D. Morris, S. Franks, H.D. Masan, M. Tuckcr élnd J. Pnee. MultifollJeular ovanes: c1imcal and endocrine features and rc~p()no.,c tn pubatlJc gonadotropin releasmg hormone. Lancet II:1375, 1 <JXS.

Adal11~, J., D. W Pobon, S Franks. Prevalenee o! polyeystlc ovane~ ln women wlth :lIlovlIlatHln and Idiopathie hm,uti~m. Britl!lh Medlcal Journal 293:353, 1986.

A~chClll1, P. ÂgHlg lT1 Ihc hypothalall1lc-hypC:1hy~cal-()varian axi~ III the rat. In: 1 Iypol ha la III li 0.,. PIIlIltary amI Aglng (A Evenll and J.A. Burges~, ed~.).

Sprlllglieid. ".' Cllilrk.., (' Thom".,. pp. 376-411-;. 1 <)7(l.

Ibhl, O.P. Ilul1ldll LllOllOlIlC gorwdotroplll: al1llllO aClù ~eqllellce o! the alpha and I)(~td o.,uhlllllh BlOc/Will. BI()phy~. RC!learch Clllllll1l1lllcatlom, 48: 146. 1972.

Blilk,~. (';\. ILl. Sc.t r,1 Jll li li 1. J IldlJard and CIl. Sdwyer. Cllculatll1g Ieveb Dt pllllilary gOIl:ldotroPIIl.., (Illd ovanan ~tcr()Jd~ ln rat~ aller hypothalamic dcalkrCIlt:ltlllll. NellI Ol'lldOCllI1ology 12:~6, 1973.

Blake. c., R. WCIIlL'r. R. Cim~kl and G Sawyer. Secretion nt pltultary luteillizlIlg hormonc and lolllclc ..,tJllllllatll1g hormone III [emale rat~ made persistently l'~t[()U~ ni the..,tl ()1I~ hy hypothalanm: deat1erentatloll. Enùocnl1ology 90:855, 1972

B()It(}lu~~I. M . G. Mafllll and Â. Dai Lago. Autoradlographlc stuLly o! the ùl~tnbutlOn 01 Lli rL'rt'plor ~ III the O\'ary ul untreatl'd ami gonadutroplIl pnmed Immature 1 :Ib Cl' Il :llld t I..,..,UC rl'..,carch 183:329, 1977

BOl 1(}lu~!-oI, M. (J, rvLlr III 1 a III 1 M 1 .. Relon. A hl~l(Jchellllcal !ltuLly 01 the bllldll1g ot Il.:''i h( 'C j lu 1 hl' rdt m dry thloughollt the c!-otrous cycle. Cel 1 and tJs~ue n'M'arch 197:213. ]lnl)

Borl(}lll~~I. M .. (J. Mal 1111, M.T. Rolandl and L. Colornho. In vitro binùing ot 1125

h('(; III thl' lat O\':lIy lrom birlh to plIberty. Cel 1 and tl~~lIe research 214:633. IlJX 1.

Brachet. J. l'hl" lurallzatlon and mie ot nhonuckic aeld in the cel!. Annual of the New York Âradcl11y ot SCience 50:X61. 1950.

74

Brawer, J.R.. F. Naftolin. J. Martin and C. Sonnescht'Ill. FI !l'cts 01 a slIlgll' \IlJl'rt Il Hl

of Estradinl VaIerate on the hypothalamlc arcuate nlll'kll~ ami on Il'prodllctlH' function m the fl'male rat. Endocrinology 103:50 l, 197H.

Brawer, J.R., H. Schlpper and F. Naftohn Ovary-dept.' ndcllt dq!l'Ill'ldtIOIl III the hypothalamic arclIate nuckus. Endocrlnology 107:27·1. Jl>XO.

Brawer, J.R., H. Schlppl'r anù B. Roham'. Elket!'. ni hlllg ICI m andlllgl'Il ,1Ill!

estradlol exposure on the hypotha\amu~. 1 ~Ild()l'nnlllllgy 112: I l ),1. !lJXJ.

Brawl'r, J.R., M. ivlunoz and R. Farookhl Dl'velopllll'nt (lI Ihe plllyl'y~tll' (lV:lII;1l1 conc.iItlon (l'CO) 111 the e~tradl()l-vakrale trl'alnl 1 <lI. BHllngy III RCl'llldllctlllll 35:647, 19~6.

Brawer, J.R.. M. H.Il'hard ,md R. Fawokhl. Pattem 01 hlllll:J1l lill li \lllllC gUIl:JdllllOplll (hCG) hmdlllF III thL' po\ycy~IIC ovary. AIl1L'f1Cilll J(lllillallli ()\l"'l'IIIl'~ a III 1

Gynecology (111 pre",,). !9X9.

Butcher, R.L.. W.E C()IIIIl~ and N.W. Fugu. P\a~l11a C()nl'l'l1tr(lt\(l1l~ (lI LI 1. I:SII, prolact\J1. proge:-.teJ olle ami e~tr(\llIol 17-hL'ta thlllllgl!()uI the -l-day l'~tmll~ cyl'll' nt the rdl. r":nùocl Ill(llogy 94: 1704. 1974.

Bysknv. A.G. Folilcular ;\trc:'Ja. 111: The Vertehrale Ovary (R.L. J(lfle .... hl). New York. Plenum Pre~~. pp. 533-562. 197K

Byskov. A.G. Atre~la. In: Ovanall 1'01liclIidr Dewl{)pme/lt and hllll'tHlil (A.R. M\(jgley and W A. Sadky, ed~.). New York' RavL'Il PIl'~'" pp ,11-57

Campbell. CS. ami N.B. Sch\vart/. The II1lpilCI 01 l'OlI~tilllt IIglIl 0/1 tht' t'\I!(JlJ" Cyl Il' 01 the rat Fnd()cflnulogy I06:12.1,(), 1 (JX().

Carither~, J.R. and .1 A. (nl'l'I\. Ullra~trlll'tlire 01 rat ()Vilflan lIltt'f~t!tJill t'db. Normal ~trllctllrL' a III 1 regre"~lvl' changc\ lulluwlllg lIypophy"l'et()llIy .JI li Il Il il 1

ot Ultia~tru('tllral RL'~l'ilf( .. h 39:2')<), \(J72a.

Carithers, J.R. and .I.A. Green. Ultfil"tructure 01 rill ()vafl.1Il IIJter~tltlill Cl'II~ II. Resp()n~e tu gonauotropll1. Journal 01 Ultril~tructllral Re~edfch )'):25 l, 1 <J72h.

Carnere, P.O., J.R. Brélwer and R. Farookill. PJtlJJt;lIy gOllddolmplll-n:il'i1 ... JlIg hormone rl'ce ptor COll te nt III ra t~ WI th polycY'·,tJC ()va fin. Blology!ll ReprouuctHlI1 3H:502. l ')XK

75

Carnere, P., R. Farookhl and J.R. Brawer. The role of aherrant hypothalamic opmterglc t unctlon ln generatmg polycy~tlc ovane~ m the rat. Canadian Journal of Phy~i()l()gy and Pharmacolo!,1)' (In pre~~), 19H9.

COl1very, M.M., (j.r. McCarthy and J.R. Brawer. Reml~~lon ot the p()lycy~tlc ovanan condl!Hm (PCO) ln the rat fol1owmg hernIovanec!omy. Anatomlca[ Record (m p re!\~) 1 <JHlJ.

('oplllilnn, T.L élnd W.C. Adéll11~. Re[atl()n~hlp of r()lycy~tlc ovanan induction to prolactlll "ecretHm: prevention of cy~t forrnatlOn hy hromocnptlfle m the rat. ElIdoCIIl1ology JOH: J (J<)), J 9~ J.

('Lllie/, M J) ,ml! ;\ Negr()-Villar. J>ubatIle follIcle-:--tllllulatmg hormone ~ecretlOn I~

IIlde 1cmfellt of lutellllllng-hormone relea~lng hormone (LHRH): pubatlie /t'platTI1lt'llt ot 1 J IRII hHI(letlvlty ln LHRII-11ll1l1l1ll0neutral!zeù rab. LlldllCIIIl<J/II!-!Y 120:2() 1 1. Iwn.

Daallc. T.A alld AI· 1',11 low. Serulll FSII and LI 1 ln con\tant IIght-lllduced per!'>l"tcnt t'"t/u .... "hOlt-tC/1ll anù long-term ~tuùle~. EnùoLrlllo\ogy 88:96 .. L \971.

Daw~(ln. A.B. and M. McCahe. The mter!'>tltlal tl!'>~lIe ot the ovary 1I1 IIlt~1 ,lUit and JlIVt'/1I1c raI<,. JOllrll.t1 01 Morphology 88:543, 1951.

Ik:lllt'.II.W. 1 II~tol'hl'/llIc.d Ch.lr:lctL'fl/atIOJ1', (lI aIrelle lolltcles III the rat's ovarres. Allal()l11lcal ReCll/ Li III :5()-l (Ahl,lral'I), 1951.

De.1I1l'. Il W. 11i,,!odll'IllICdl llh\l'f\'atloll:-' on the ovary and oVlduct 01 the alhillo rat dU/l/lg thl' c"t/Oll\ lyl·lL'. AI1ll'f1ca/1 J(}urnal of Anatomy 91:,3(),3, 1952.

Ikanc\lv. R ()/lgII1\ and dt'\elllplllL'nt llt IIltl'r~lItl,t1 tl\~Ue III ()vane~ of rahhlt and gllllll"l-plg JUlIlll,d 01 ,\ndlo/llV 113:251. IlJ72

1 klllp~t·Y. 1· W. R () (;/ t'cp and Il W DL'.Ille Change:-- III the dl!'>tI Iblltlon élnù WIll't·llt/.IIIlI!l (lI .t1kalllll' ph(l~plldta~t.'\ 1/1 11~~lIe" (lI the rat alter hyp()ph~~l'l't(1llly ()J ~()nadt.'ctul1ly and altc/ replacement therapy. Ellllocnnology 44:Xs' 1l)..llJ.

Dhol1l. (i. and lU. Ml'nde. DIl.: alkalI:-che phl)~rhata"e III den hlluszelkn des ovars. VllchllW'~ Arch. 328:,337. 1956.

DIL'I'M·Ill'. D .. A.N. Battadwryd. L.E. Atkll1:-.on and E. KnohJl. ClrcllOral o~crllatlons (lI pl.l~llla 1.I1 kve b III the ovanectUllllzed rhel-lI~ monkey. Endocnnology X7:X50. 1970.

76

Dlmino, M.J. Dlfference~ m mitochonùnal ~tt'nlJ(logt'nesl~ hetwt'l'11 tolhelll.1I and luteal ti~~ues of porcine l1varit'~. EnùnCflllology 101: 1 S4.f. 11>77.

Dimmo, M.J. and K. Herman. Increast'~ III mJtol"llOndllal ~tl'II)Jdllgl'Ill'~I' alter ~Ilolt term II1cuhatlOll of porc 111 t' ovanan tol1ldl'~ Wlt h lutl'lIll/lIlg hm nHlI1l'. Endocnnology 104:7~O, 1979.

Dufau, M.L.. K.J. Catt and T. T~lIrllhara. Blologleal actlvlty ot hlll11i1n dHllHlIlIr gonaùotropm reka'l'ù trom tl'~tl\ hmdlllg ~Itl'~. PIOCLTdll1g\ ot the N,Itlllllal Acaùemy ot SCIence 69:2414. 1972.

Engel, L.L. The hlmyntht'~I!\ ot l'\tIogl'Il!\. In: Ilallllhook 01 Plly\IO!tlgy-Endocrll1olo!:,Tj' Il, Part 1 (R.O (;rccp. t'(!.). BaItIIlHHl', MdIY\;)IIlL Wawllv Pre~s. pp. 4()7. 19D.

Encbol1. G.F. Pnlll;IIY cultuIl'\ of ovallall l'db 111 ~l'llIl1l-lrl'l' Illl'tlllll1l iI\ nHllll'b ot horJ1lllI1l'-Ùepelllknt thlklclltlatlllll. MllkeuIar a III 1 ('l'lllIlal 1 :ntlol'lllllliogy 29:21. Il)~J

Encbnll, G,r. D,A Magllttlll, C.A. Dyer a III 1 ( Illlkdltl 'l'hl' ()V;)II.l1l allt!lllgl'II produclllg cl'Ih' a rl'Vll'W 01 ~trllcture/tllllrtl()11 1l'I.ltll)Il,llIP\ 1',lldl)l'IIIll' RI'VII'W\ 6(3):37 I. 19S:\

E~hkol. A. and B Lullt'llklLi. Cionm!otropil Il'glllatlllll 01 (lv.lII;III dl'wl()PIIll'l1t 111

mlCl' dUrlng IIllallly. 111' (Jonadotr()pln\ (B B. S.lVl'IW. ('J; IklJJlg ;IIH! Il M Gralldy, l'd\.) Ne\\ York. John Wlley ami ~()m, IIlL. pp n')- lll) Il>72

Farookhl, R .. R 11c1l11l111lg~ alld JR Br;lwer lhlll<ltl'lal ()Vcllll'lt())lIy Il'\tlllt'\ ovulatory cyellwy III rah wlth a 1}()lycy\lll' ()V;lllall lllmlJt)())1 H)(ll()gy III Reprod uct Il)Jl 32:~_"'(). Il>X~

Pawcett. D.W , J.A. Lllllg .11](.\ Â L. JOlIl'\. '1 III 1IItr~l\tI1l('tllll' 01 tilt.' l'lId()('f)IIl' glaJld\ Recèllt Prp,L!fl'\\ 111 Ilmmllne RC\l'dnl1 25:1,1::;. I%l)

FlIlk. G. reedhack actllln\ 01 targct hornHlnl'\ on thl' hypotl];ll.llIIlI\ amI pltlllt;lIy wltl! ~peclal rderellcc t() gOlladal !\tcrllld\. ÂIlI1U;t! Revlcw ()I l'hy\l(d()gy 4):~71, 197<)

Ford, D.H. ami A. IIlr!'>chl1léln. AlkalmL' Plll>\ph~lt;l\l' aLtlvlty III tlll' ()vam'\ of Immature and matunng alblrlo rat!'>. Allatorlllc;J! Record 121:')31, Il)')')

77

Fortune, J.E. and D.R. Arm~trong. Hormonal control of 17-beta-e!o.tradlOl bJOsynthesis m proe:-.trou\ rat jolhcle!o.: e:-.tradiol production by i:;olated theca ver<;us granulma. J~nd()cflnolobry 102:227, 197ft

Franchmont, P. A ~tlloy 01 the crm~-reactl()n hetween human chononic gonadotronin and pltultary lutelfll7Ing horm()ne~ (hCG and hCG). European Journal ot ClJmc;t! Jnve~tlgatlon 1 :h5, 1470.

(jallo, R V. amI A Bona-Gallo I,ack nI OVélflan sterOld negatlve feedhack on PlIl\;ltlle IlItl'lIl1zlllg hmrl10ne reJeml/lg hormone hetween e~troll:-' and dle~trou~ day 1 III the r.lt e~tf()lI:-' cycle Endomnology 116: 1525. 19R5.

(jold/leher, J W .1Ild l, R Axelrod. Clmlcal ,Ind hlochemlcal teatllre:-. of polycystic OVélllélll dl\l'ac.,e hrllllty and StenlJly 14:631. J 963.

(jold/ll'hl'1. J W »()Iyly\tll ()V.lrJan dlc.,l·él~e. FntJllly and Steflltty 35:371, 1<)~1.

(ior..,kl. RA (ionadéll IHlInHlIW\ .lI1d the perlfléltal development 01 neuroendocrine IUIlClllllL III l'lolltll'fc., III Neuroemlocnl1()logy (L. Martini alld W.F. Ganol1g, l'th.) New York (hforL! lJnlwr'dty Pn.~c.,c., pp 237-2lJ(). 1%9.

(il()c.,c.,l'J, PM., (i,F MC(';llthy. B. Rohaire, R. Farookhl and J.R Brawer. Pla:-.ma patternc., uf LI L (·SII ,lIld pllllactll1 111 rat ... \\llh a plllycy ... tlC oV;lnan conditIon IIldul'nl hy l'!'.!léldllli VélkLIIl', Endocllflolugy 44:3J. IlJX7.

(illlélya. S S éllld (j S. (ill'l'Il\v.lld A comparative ImtocheJ11lcal !'.tlluy nt II1ter:-.tltJaI Il''''lIl' ;llld f()lllclIléir atrec.,la III the 111:tll1ll1ahan o\'.lry. Anato!11lcal Record 149>lll. I%S,

11.11111. J) W alld.ll 1\1C<illlle. The andJ()gen :-.tenllz~d filt IIlOUCIHlI1 ul ovulatIOn alld 1I11pl,lIItallOIl Ily 11Itelllllll1g IHlIl1HlI1e relea~lf1g horlllone. Endocflnolo!:,'Y 102: 1741. l(nS

Iléll,I:--/. B 'l'hl' l'1Il!lll'lllll' l'Ikl'b (lf l:-.olat)()Jl nt the hyputh:llélllll\'l Iwm the re:-.t 01 thl' hlélill III h()lltll'lc., III NèllroellL!ocnnology (L. Martlfll and W.F. Ganong. L'd!'..), New Y(H)..: (hl()fL! lJlll\'cr:--lty Pre:-.:-.. p, .~(Î7. 1%9.

1 \an, S.S., lU. Ibpl1Il'llll, M 1. Chu, AN. Hm,htlL'ld ano AR. Mldgley Jr. (ionadotroplIl rel'epl()[~ III rat ovaflan IJ:-.:-.ue. Il, Suhcellular localtzatllll1 nt LH hllldlllg :-.Jtl'~ b} l ~M fllLltoillltngraphy. Enùncnnology 95:)fN, 1974.

7H

Hemmings, R., R. Farookhl and J.R. Brawer. Pitllltary and OV;IIIi11l rL'~pon~t'~ tll luteimzing hormone releaslflg hormone ln a rat \VIth plllyl'y!'>tIC (l\'allt'~. BI( Jiu!!\' of Reproùuctlon 29:239. 19K3

Hillier, S.G. anù G T. Rm~. Eftecl~ ot t':\llgt'110US tt'~to~tl'ronL' on ll\-.III.111 wl'Ight. follicular morpholo!,'Y and IIltraovanall proge~ll'r()Jll." l'1l1l1'l'Jltr.IIH1J} III nlrogt'Il­primcd hypophy~ectllm Ized 1111111<1 t lire ft> male ra t~. Blolngy 01 Re plodlll·t 1011 20:261. 1979.

Ireland, J.J. and J.5. Rlchard~ ;\ prevlomly uIH.k~lTIl)L'd Il)\l' Illl llill'lIlI/lllg IHlIllHlIll' (LH:hCG) Oll lolhcular ccII dllkrentlatloll \:11l10Clllllllogy 102:\·l)~\. \\)7K

Jacohy, F. Ovanan 111~t()chel11l~try ln: l'Ill' (h-ary. Vol li 111 l' 1 (S. IUl "t'Iman. I-t! ) New York' Academlc Pre:--:--. pp PN-245. 1%2

Kammerman. Sand R.L ('anill'id Iïlt' 11111111111(;11 01 hllldlllg III IlHIIII.ltl'd 1111111.\11 ChOf!OIlIC glll1;ll!otroplIl tn nlllll,>e ()valy 111 Vilio. , ':Ilt!()C! lIlulugy (X): 'H-I. 1(172

Kammerman. S .. CL Call/leld. J. Ko!ella and CP ('IJ.trlllJrlg. 'l'Ill' 11Ifllllllg ,,/

jodll1ated hCC! 10 porclI1e gl"lIl1lo~a l'elb. l'I1lI11l'lIll()lugy 91:11\ 1(J72

Karn()v~ky, M.J. A lorll1akkhyù~-gllltaraldehyd~ II\allve (lI 11Igh o:-'Illolallty \tH Il'>t' III, - electroll mICf()~Copy Journ;i1 ot Cl'Il Blology 27: lJ7.1. 1 ()()~

Karnov~ky. M.J. lhe (JI krrnl'yalllde-Icduced O'>lllllllll letrw,ldt' 111 elel'IIOII I11ll'lU~C()py pJ(lcet."dlllg~ nf thL' 14th Ânl1l1al Mt'l'tlllg 01 the Âlllt'Ill'all SO('Jl'ty

ot Cell Blu\ogy, Il:I·I(I, 1 (n 1

Kawakaml. M. ,1Ill! "l VI"('WI\ .IIi Thl' dtlkllllg rnplllI'>IVl'IIl'''''' 1 II thl' 11lIddk-anlerl'lI hYjloth;t1.!IllIl' IIICd to e'>1radHll Ill'II/();lIl' lI11pl;lIlt cOl11pell'>t1toly ()Van;lIl hypt'llmphy 1-.lld()('JIIl(II()gy 7-1:::'71. l'n')

.lIlll'l H II .111<1

11l11l1l11J(11l (lI

Klng~hLlry, B.I' Atre'>l.! .Jlld Ih~ IlItl'r,>tltliti cl'II'> ni tl1l' ()V;IIY ÂllIl'lll'dll .IulIIlI.II (lI

Alléltllll1V 65.'(l(J. l'JYJ

Kent, J. 50llle Imtol'hL'l11lcal ct1<l1lge~ 111 Illllllatun: IlHlll'>~ O\'dll~'" altel g( 1Ililll(ltrIJjllll

treéltlllL'nt. Journ,t1 01 EnL!ocrrnology 61:xXI, 1974.

Kent, J. anù M Ryle. 111~t{)cheIllICal ~tudJ(.~~ 011 tllr~e g()l1a(/(llroplll-rt''>jl()l1\lVl' enzyrne~ III the mtantJle m()u~e oVilry_ Journal ot Reproductlo/l ,lIltl h'r t !lJly 42:519, 1975.

7<)

Khoury, S.A, N.E. Reame, R. Keleh and J.c. Mar~hall DIUrnal pattern~ of pulsatile lutemlZlng hormone ~ecretl<m In hypothalamlc arnenorrhea: reproduclhlllty and re~p(jn~c~ to oplate blockade and an alpha-adrenerglc agom:-.t. Journal of Chlm:al f:ndoCflnology and Metabol1:-.m 64:755, 1lJR7.

Knlgge, K. and i Lcathdlll. Growth and atre:-.m ot tolltcle:-. In the ovary of the h;II11.,tt:r. Ana t< 1I1l1e; tI Rccord 124:67<), IlJ54.

KIlOhd, f:. 'l'hl' nellrllClId()l'flllC control ot the men:-.trual cycle. Recent Progres:-. in 1 J()rIllOllC Rc..,carch 36:53, 1 <)RO

K()ch. Y., V. Zor, P Choh!'.ICllg ct al. BIIldlllg ot lutemlzmg hormone and human d1<)f1()llll gOI1i1dotmplll lu ovanan l'l'lb and actIvatIon 01 adl'nylate cycla:-.e. New l·ng1.lJ1d JOllfll,tI 01 Medlclnc 29]:]79, ]974.

K()pllw.!, lk.ltn\ M A COll1p.lfl',OI1 (lI van<HI" pIOcedlllc:-, lor IlIle gralll dcvelopment III ekellOI1 11l11l1)\l'OpIC radlo(JlItogr.lphy. 1 1 1!'.lochl'lllI:-.try 44:20 ]-22'+, ]975.

K( llH I\\'a, B M, (i l\1 I.c\ 1 Ill' ,1Ill! N J. Nadlcr. A.,..,c..,\lllellt 01 re!'.ollltioll by hait dl:-.taIlCl' \ ;tllIC\ lor Illtllllll lInd 1 m!tmodll1c 111 c\ectron 111Icrmcopic ladJ(ldlltogrdph:-. ll''IlIlg IlIorLl L.+ clllublun dcvelopcd by ":-.odlum plly\lcal" or D­l<Jh Illl'Ihml., 111\lmhl'llll\try RO:519, I<)H-t

1 l'l', (' y ;lIld R.J. Rv.tll J'hc centntug,d tractlollilllOll of [125 Iabclkd human lutCIIlI/lIlg hm 1lI0\ll' (111.11) accull1ulated hy rat ovaflan :-.ltce:-.. In: Protelll and l'lllypl'plllk IIOflllO!ll'" (1'.1 !\1argoulle., and F.C. Greenwood. elh.) L\elpta Melllc.!, Armll'rdillll p, 513. 1972,1.

1 t'l', (' y ,Illd R J. R);IIl. 1.1IIl'lI1l/rng horlllonc rccèplur:-.. Speclflc hllldll1g ot hUl11an 1 Il tll hlllllllgl'IIi1ll'" ut 11Ilelflli'cd rat marre!'. Procèedlng~ ot Ihe Natlollal Al,ldt'lllv of Slll'IH'l' (,9: ti20, IlJ72h

1 crdclllK'IgcI, )' .1 III 1 L r. RClchert, .Ir. Evaluation ot a rat le!'.tI:-. hOlllogcnate Iiidloiigilllli [L'Cl'ptlll a!'.~i1y tOI' hUl1lan pllllllary LI!. EndoCflllolugy 90:3H.+, 1972.

1 PlI\l'I . .1 P .. S.M Il.trlllall, l.R. Sl'hrl'Iher dnù G T. Rm:-.. EVIllenl'e for a role ot .1 11 lit ngl'll\ 111 Illlhl'Ular maturatlOIl. EmloCfmo]ogy 97:3()(), ]l)75.

1 1Illl'llltl'ld. B. and A. hhklll. ImmuI1u\ugy ot human Chorh)I1IC gonaùotruplll (hCG). VIt. Illlfllm!lC 25:137, 1%7.

RO

McCarthy. G.F., J.R. Brawer and R. Famokhl. Pla~ma III p:lttl'lll~ ch.llactl'll/lllg thl' develnpment ot the polycystic ovanan comhtloll (peO) III thl' l'~tl.ldllll valelatl' (EV)-treated rat. Blolob'Y of Reproduction 34 (suppl. 1):1)3, 1')l"ih.

McCarthy, G.F., R. Farookhl and J.R Brawer respnn~e to a ~peCltJc pubatlle pattern ot of Reproductloll 36 (suppl. 1):172, !9X7.

pl)I\'l'\'~tll' l)\'illll'~ (PC'()) lll'l'UI III

lutl'llll/lIlg IHlIllllllll' (III). Bllllllgy

McCarthy, G.F. and J.R. Brawcr. Inductloll ot a StclIl-I.l'\l'llthilt tvpe 1'1)1V!'v\tlc ovanal1 condition (peO) Jf1 the rat: a new lIlodet lUI rv\tlc llvallilll dl\I'i1\l' (Suhlllitted), IlJSlJ.

McEwen, B.S. Sterold hormone IIlteractlOIl\ wlth thl' hl""!. n'l\lIl,1I dlld lllllkrulill a~pect\. In Rcvlew~ ot NeurmCIence (D.M SChlll'ldl'l. l'lI) New Ylllk Ravl'n Prc~\. pp. 1-30 !()7t).

McKay, D.G .. J.II M. PlIll...clton. A r. 1 krtlg and S D.lI1/1gcI. ï Ill' ildult hU1ll.l1I ovary' a hl~tuchel11lcal ... tudy. ()h~tl'tm'''' ami C'ylll'lOl!lgy IX:I '. Il)() 1

McNatty, K P, A Mak[J~. (J. DeC,ril/lil, I{ O'o.ltl1iIIHlllh, illlli K.I. I{Yilll. 'l'hl' productl()n ut proge~tl'rone. andlogl'Il\ :llld l''otl()gl'Il'" hy glilllltlmii l'l'lb, theral tl~~lIe and .... roll1al tJ ... ~ut.: trom 11l1llldl1 OVtlrIl'~ 111 vItro J(llllllilllli ('1llllCiil EndocrJnology amI Metah()II~1ll 49:()K7, 1<)79.

Mahe~h, V.B. Currt.:llt COllCt.:ph 01 thl' piithophY"'I()lllgy (Jt titI' pOlyly\tll llV.IlY ~yndrome. In. Endocnne PhY"'Hljlathol()gy (JI the ()vilry (R 1. ')'()/lllll, (j Rl·t·Vl· ... and R L. Pmeda, l'LI .... ). Am:--terdalll: I.I:-.evll'r/N()lth-II()II.1lld BUlllIl'dll.d l'Il· ....... pp. 27)-2<)4. 1 <)i)().

Mahe~h, V.B. and RB. Greenhlatt. Stl'flllll ... t.:cll'11<1I1\ III thL' lI(lIlllid .lIld p()lyl'y~tll'

ovary. Recl'nt Progre·,., III IlorlllOlll' Rl'l.,l'.lIch 20:,·11, 11)(,,1

Malone. TL Oh:-.er\'dtIl lll... \lll the glycerophmphillêl"l' 111 devellljJlng Journal (lI Aniltol11y 106:41, 1 ()(j()

hl:-.tochellllliil Im;i1I/i1tlllll lorpold lutl'il 01 ;lIhlllll I.lt\

01 :lIkalllll' ÂlIll'( Il ;111

Matll~(), H., Y. Baba, R.M (i. NalL A Arllllllr.l dllll A V. '>lh;tlly '>tluctule (lI the porclI1e LI 1 and FSII ;-t.:lea~lIlg horlllOlll' 1 he P/()jl()..,l·d il III IIJ( 1 .lt'ld ... l·ljlll·IICl· Blophy~IOI()gy Rl'.,earch COll1IllUnlcatloll'" 43: 1144. lin 1.

AI-Mehdl, M.J.H, hne ~truclure and llngm 01 Intel ... tltlill lI ... ..,ue ln the llvmy 01 the

pregnant rat. Acta Anatollllca 103:245, llJ7<).

Mcyer, R.K. and W JI McShan. Enzymes in the Ovarian Tb),ues of Rats. In: Mcn.,truatlon and Ih Disorder~ (Earl Engle, Ed.). Springfield, IL: c.c. Th()ma~, pp. 62-<)5, 1950.

MH.lgley, 1\.R. RadlolmlllUn()a~~ay: a method tor human choriomc gonadotropin and hllman 11IkJnlZlng hormone. Endncnnniogy 79:10, lQ66.

MH.lglcy,1\.R ÂlltoraulC)graphlc analy~ls of gonadotropll1 hmdmg to rat ovarian ti~sue .,CCtl()lI~ Âdvancl'~ ln Expenmental Medical BJ()lob')' 36:365, 1973.

Mldglcy, 1\.R , Â J Zl'le/nlk. J.S Rlcharu~ and S.S. 1 Jan. Gonadotropm and e~tradio\ rcn:ptor~ and flllillular uevelopmcnt. In: Advance~ III Fl'rtllIty RegulatIOn through Ba~lc RL'~l'.trch (W A. Sad/er and 5 Segal, l'ès.). 197..t.

Mohh~, C'. V .. K FllII key. D.M Gee. K Y,IIll<IIllOlo. Y.N. Sinha and c.E. Flllch. blr;ldl(JI-llllllllCd :Idult anovulatory ~ynJf{)ll1e ln femak C57bl/6J 11lIce: age-like IIl'urm'IIt!(Jl'I me. hu: nol ovanan. Illlpalrll1ent~. Blolob')' of Reproduction 30:556, \lJr;4

Morgan, F.J. amI R I:. Canflcld. Nature of the ~ubumt~ ot human chorionic gllllal!()lropll1. Ellllocnllology SR: \ 045. 1971.

Mmgan, F.J .• R.E Callfteld. lL. VaJtLlkaltl~, anù G.T. Ro~~. Propertie~ ot the ~lIhllnll~ (Jf hll/llan Chorl0l11C gonadotropin. Endocnnology 94:1601, 1974.

MOrl, II. alld K. Mat~lIIlHJtO. Dcvelopment of the ~ec()nuary inter~titial gland in the rahhll m ary J Ollf Il a 1 ()f Alla tOll1y J] 6:0.117. 197.3

Nll1lrod, A. Ci F Lncboll and K.J. Ryan. A ~pecitlc FSH receptor in rat granulma l'l'II~: PlopLllle~ of hllHJlng ln vitro Endocrmology 98:50.1974.

82

Omura, T., E. Sanders, R.W. Estahrook, D.Y. Cooper ami O. RllSt'nthal. hlllattoll from adrenal cortex of a nnnhemc mHl protelll and a tla\'ol pllltt' III IUllctlolllll as a redllced triph()~phopyndine nul'lt'ltlde-cytochwl11t' P-.t:'O ll'llllrta!'>l'. ArchIves nt Blochemistry and Btophyslology 138: 16·l-IHO. 1 %h.

Parker, C.R. and V.B. Mahe~h. Interrelatlon~hlp hetWl'l'1l t'\Cl'S!'>IVt' kVl'b III circulatmg androgen!'> ln hlond and oVlJlatory Iiltlurc. Journal 01 Repllldul'tl\'l' Medicine 17:75, 1976.

Payne, R.W., A.A. Hellballll1 and J.N. Owell!'>. The etlert 01 amlrllgcll!'> Ull tlll' UV. Il Il'' and lIterll~ 01 the e~trogen treatel! hypophY!'>l'Clllllll/cd 1111 Illat III l' rat. Endocnnolngy 59:J()(l, 1 ()56.

Payne, R.W. and R.l1. Rlln~L'r. The 1llli1lenCe 01 l'~tlOgell a III 1 alldrogl'11 Oll thl' ovana n re~p()Il~L' 01 hypllPhy~el'tOl1lIZed 1111111:1 t III l' 1.1 h tll g( li .adot t 0p"I'. Endocnllology 62:J!J, 1 ()5X

Peder~()ll, T. FoIIIC!e kIllCIlC\ 1I1 the ovary 01 the cydir 1l101l\l'. Arta Flldlll'l Plllh 19l1'.1 64:304, 1970.

Peluso, J.J .. C. Engla(1d-(,hark~Wl)(th D.L. Boknder and RW. Slegl'1. lJltla~tllll'llllal

alteratl()n~ a~~()clatl'd \VIth the II1ltlation 01 fllllicular atre~la. ('l'II and TI\\lIl' Resea rc h 211: 105, 19XO.

Peter~. H. The developllll'Ilt 01 the ll1()lI~e ovary Irom l))Ith to lI1atullty. Ârta Endocnilologlca 62:()K 19h()

Pinkerton, J H.M, D.G. McKay and E.C. ;\ùal11~. Developllleilt 01 the hUllIall ()vary­a ~tudy u~lI1g Imt(}clH:'llllcal tèchlllqUl'-'. ()h\tl"tl'lc~ and (;YI1l'l'()I()gy IX: )52, I()f,)

Ponder, B A and M.M \Vtlklll\(lll. InllihltHlI1 01 l'I1t!OW'1I01l" tl\\lIl' alk.dllll· phmphaul\l' c()IlJugatL'\ III Illll11unohl\t()chl'll1lt.,try J()lIIIl.iI (lI Il J\t()('IIl'IIlI\tly and CytoChlllll\ t l'y 29: t )K l, IlJK 1.

Popkm, R.M., H.M. f ... a~cr dnlÎ Re;. (J{)\dl'Il l-jtc('t~ 01 1 J IRII-.Intdg(lIJ\\t (lll 1./ f RII lI11munOllelltral1zatHlIl on pltllltary alld ()Van;11I 1.lIRII rl'tl'plO/\ III It'lIli1k r ;Il~. Journal 01 ReproductIon éllld Fertlllty 6<):24\ 1()Xl

Rajamemi, J. and T. Vanha-Perttula. SpecifIe rl'l'l'ptor lOI LI 1 III tilt' ()v;lry. l VldCIII'l' hy raù loa utogra phy anù tl~~lIe t féll·t J( lIla t J( ll1 LnL!()Cflllo){)gy 90:), 1 ()72

H3

Rajanicml, lH., AR. Midgley, J.A Duncan and L.E. Reichert. Gonadotropin reccptors in rat ovarian tissue. III. Binding sites for luteinizing hormone and ditfcrentléltlOn of granulosa cells to luteal cells. EndocrinolobT)' 101 :898, 1977.

Rajilnlcml, J II., AN. 1 Ilr~hfleld and AR. Midgley JI. Gonadotropm receptors in rat ovanan tJ~~ue. l. LocahzatlOn of LH hinding ~iles hy fractlonatlon of ~llhcelllliar ()fganeIlc~. Endocnnology 95:579, 1974.

Ramcy, J.W., R.F. Hlgh~mlth, W.W. Wilfmger and D.I\A. Baldwin. The effects of gonaclotropin-relea!>mg hormone and estradiol on luteinizing hormone himynthe~l" ln culturcd rat antcrior pltultary cells. Endocrinology 120:1503, ] 9~7.

Rt'bar, R.W., II.L. Judd, S.S.c. Yen, J. Rakoff, G. Brandenherg and F. Naftolin. Charactcnzatlon of the mappropnate gonadotropm secretion m polycystlc ovary !'.yndroll1c. Journal of Ciimcal Inve~tlgation 57:1320, 1976.

Rt'l1llcls, E.G. Influence of hormones on the hl~tochemi~try of ovarian interstitial tl~~ue III tht' 1I11111ature rat. Amcncan Journal of Antomy 88:63, ]951.

RICC, B.F. and K. Savard. StCfOld hormone formation in the human ovary. IV: ()v:IrI:1n ~tromal cOlllpartment: formatIon of radIoactIve sterOld~ from acetate­I_J.l(' and action ot gonadotropms. Journal of C11l1ical EnùocrmolohT)' and Meli1h()II~1l1 26:593, 1966.

Richard, M:II tille. Locallzation of LH/hCG hlllding sites III the polycystlc ovary. A the~l~ !'Iublllitted to the Faculty of graduate Studies and Research in partial fuIl III111cnt of the rcqlllfl'ments for the degree of Mmter of Science, McGill LJI1lVl'r~lty. MOl1treal. Octoher 199B,

Rlchard~, J.S .. J,J. Ireland, M.C. Rao, G.A. Bernath, AR. Midgley and L.E. Reichert. Ovaflan tolhcular development m the rat: hormone receptor regulation by e~tr:Jdr()l, follJcle ~trmlllatmg hormone and luteinizmg hormone. Endocrmology ')9: 1562. 1976.

Rlrhards, 1.S. and AR. Miùgley. Protem hormonal actIOn: a key ta undentanding ovaflall folhclliar (lnù Illteal œil ùevelopment. Biology of Reproduction 14:82, 1976.

Rohaire. 8., I .. L. EWlI1g, D.C. Irhy and C. Desjardins. Interactions of testosterone and estradlol 17-B on the reproductive tract of the male rat. Biology of Reproduction 21:455, 1979.

84

Rohaire, B. and B.F. Hales. Regulation of epididymal glutathione S-t! iln~ferasl's: effects on orchidectomy and androgen replacement. Biology of Reproduction 26:559-565 1982.

Ross, G.T. Gonadotropins and preantral fnllicular matUIation in wumen. Fcrtihtyand Sterility 25(6):522, 1974.

Rowe, M.J., R.R. Bieszczad, M.A. Neymark and M.J. Dimino. Synthcsis of Mitochondrial proteins after stimulation of ovarian fnllidc:. by lutl'Illillng hormone. EndocrinolobY)' 108:127, 1981.

Rowe, M.J. and J.L. Hopko. Luteinization-specif~c ovanan mitochondnal proteins. BiolohY)' of Reproduction 35:906, 1986.

Ryan, K.J. and O. W. Smith. Biogencsis of estrogens hy the human ovary. IV. Formation of neutral steroid intermediates. Journal nt Blologleal CIlL'l1li~try 236:2207, 1961.

Ryan, K.J. and Z. Petro. Steroid biosynthesis by human ovarian gfé111ulma and thecal cells. Journal of Clinical Endocrinoloh'Y and Mctaholism 26:46, 1%(1.

Ryan, R.J. and c.y. Lee. The role of membrane bouilli reccptors. B\()logy of Reproduction 14:16, 1976.

SchuIster, A., R. Farookhi and J.R. Brawer. Polycy~tic ovanall colllht!OIl III estradHlI­valerate treated rat~: ~ponté.lneous changes 111 charactenst.c elll!oCf1ne leatlllcs. Bio]ogy of Reproduction 31:5H7, 1984.

Schwall, R. and G.F. Erickson. FunctÎonal and mo:-phologlcal ché\ngc~ III rat theca cells during aLresia. In: Dynamics of Ovarian Functioll (W.B. Schwartz and Hunzicker-Dunn, eds.). Raven Press, NY. p. 29. 19S J.

Shaha, C. and G.S. Greenwald. Autoradiographlc anaJy~is 01 changes in ()Vélllan

binding of FSH and hCG during induced folhcular atresJa in the haJll~ter.

Journal of ReproductIOn and Fertllity 66:197, 19H2.

Simard, M., J.R. Brawer and R. Farookhi. A'l IIltractabk, ovary-lI1depc/ltknt impairment in hypothalamo-pituitary function in the estra(II()/ valeratc-Înduœd polycystic ovarian condItIon III the rat. Bloloh'Y of Rcproductlon 36:1229, 1 <JX7.

Stafford, R.a., W.H. McShan and R.E. Meyer. Acid and alkallllc ph()~phatrl:-.es lTl

ovarian tissues of the rat during pregnancy and lactatIOn. Endocflllo)ogy 41:45, 1947.

S5

Stratton, L.G., L.L. Ewing, D.C. Irby and C. Desjardins. Effieacy of Il'stOStl'IOI1C­

tllled polydimethyl-siloxane implants in maintaining plasma testostcmnc in rabbits. Journal of Reproduction and Fertility 35:235, 1973.

Uilenbroek, 1. Th. J. and J.S. Richards. Ovanan follicular devdopll1cnt during the rat estrous cycle: gonadotropin receptors and follicular responsivcnl'ss. Biology of Reproduction 20:1159, 1979.

Uilenbroek, 1. Th. J., P.J.A. Woutersen and P. Van der Schoot. Atrl'sia of preovulatory follicles: Gonadotropin bindmg and stcrO\ùogl'nic activlty. BIlllogy of Reproduction 23:219, 1980.

Vitale, R., D.W. Fawcett and M. Dym. The normal dcvclopment of the blood-tcstls barrier and the effects of clomiphene and estrogcn trl'atment. Anatol1lical Record 176:333, 1973.

Waldstreicher, J., N.F. Santoro, J.E. Hall, M. FIlIcofl ilnd W.F. C/Owlcy. Hyperfunction of the hypothalamic-pituitary axis in womcn wlth polyryst ic ovarian disease: indirect cvidence for partial gonat!otropin dcscnsitil'iltioJ}. Journal of Clmical Endocrinology and Metabollsm 66: 165, 1 <)gft

Wildt, L. and G. Leyendecker. Inùuction of ovulatIon hy chrome admllllstratlot1 of na1trexone in hypothalamic amenorrhca. Journal of Clmical Endocrinologyand Metabolism 64:1334, 1987.

Wilkinson, M., R. Bhanot and J.R. Brawer. Prolonged cstrogcn treatmcnt IJ1dUCl;S

changes in opiate benzodiazepine and beta-adrenergic hlllding ~ite!-. III fcmalc rat hypothalamus. Brain Research Bulletin 11 :279, 19X3.

Wilkinson, M) R. Bhanot, D. Wilkinson and J.R. Brawer. Prolonged c'ItlOgen treatment induces chances in opiate, henzodiazcpll1e and heta-adrencrgic binding sites in female rat hypothalamus. Brain Rcsearch Bullctm 11 :279, I<Ji-O.

Willdnson, M., J.R. Brawer and D. WIlkinson. Gonadal steroid-induced modification of opiate binding sites in anterioï hypothalamu~ of temaJc rat~. BI%gy of Reproduction 32:501, 1985.

Yen, S.S.c. The polycystic ovary syndrome. Clmical Endocrin%gy 12:177, lc)HO.

Zeleznik, A.J., A.R. Midg!ey and L.E. Reichert. Granulosa ccII maturatIon Hl the rat: increased binding of human choriofllc gonadotropin following trcatmcnt with follicle stimulating hormone in VIVO. Endocrino!o!:,1)' 95:H 1 H, 1974.

86

,

FIGURE 1

FIGURE 2

FIGURE 3

FIGURE 4

Light micrograph of a polycystic ovary 56 days after EV-injection. There are no corpora lutea and few secondary foilicies. Precystic (pc) and cystic (C) foilicies are a prominent feature of these ovaries.

xl3.5

Light micrograph of a Type III Large Foilicular structure (III) from an EV-induced polycystic ovary, next to a macrocystic follicie (C). Note the large size of this structure relative to the cystic follicle, and the differences in the structure of the membrana granulosa and thecal wreaths.

x90

Light micrograph of an EV inj ected polycystic ovary. Three interstitiai cell clusters (c) are Iabelled. A portion of a cyst wall can be seen at the top of the field.

x90

Light micrograph of an Ez-induced polycystic ovary. Clusters of secondary interstitial cells, engorged with lipid, are particularly abundant in these ovaries. Severai clusters are located in the upper left-hand side of the field. The size of the microcystic foilicie (c) present in this field can be compared ta the macrocystic foilicie in figure 2, photographed at the same magnificatian.

x90

•

•

, ,

" J fil . '1' ~.

, ....... " :.'

\ C

TABLE 1 Surnrnary of the:. relative expression of alkaline phosphatase following irnrnunostaining with biotinylated antibody against alkaline phosphatase in various cells of cystic and non­cystic ovaries.

In the rating scale used to index alkaline phospha tase express ion l "0 Il den otes no act i vi ty , "±" an occasional posi ti ve reaction, "( +) Il a weak reaction; "+" a cons istent and def ini ti ve positive reaction, and "++" a strong reaction. Because vascular endothelial cells of the rat ovary show considerable alkaline phosphata se activ i ty, an asterisk is used to signi fy v'hen at least part of the reaction observed for a particular structure is qiven by blood vessels.

TABLE l.

Relative Expression of Alkaline Phosphatase following Immunostaining with Biotinylated Antibody against Alkaline Phosphatase

in various cells of cystic and non-cystic ovaries

FOLLICLE AND CELL TYPE

PRIMORDIAL/PRIMARY -granulosa -thecal

SECONDARY/GRAAFIAN -granulosa -thecal

ATRETIC -granulosa -thecal

PRECYSTIC -granulosa -thecal

CYSTIC -granulosa -thecal

SECONDARY INTER­STITIAL CELLS

CONTROL OVARIES

(+)

++*

++*

NIA NIA

NIA NIA

+*

EV-TREATED OVARIES

(+ )

++*

++*

++*

(+)*

(±) *

E2-IMPLANTED OVARI!=:S

(+ )

++*

++*

NIA NIA

(+)*

(+) and ++*

FIGURE 5

FIGURE 6

FIGURE 7

FIGURE 8

Light micrograph of a control ovary stained for alkaline phosphatase (AP). Immunostain reaction product dppears black in these micrographs. AP activity in stromal cells of primordial foll icles (arrow) is weak compared to AP activity in prirnary (small arrowhead) and secondary (large arrowhead) follicular theca.

x225

Light micrograph of a control ovary stained for AP, illustrating relative intensities of AP activity in ovarian structures. AP activi ty is most pronounced in secondary follicular theca (h) .

x13.5

Light micrograph of thecal cell layer from a secondary follicle (h) and a microcystic folljcle (c) stained for AP. Endothelial cells are indicated by arrows. Note the major difference in thecal cell sta ining between the cyst and secondary follicle.

x225

Light micrograph of a control ovary staincd for AP. The thecal reaction of heal thy antral follicles (h) to AP irnrnunostain is comparable to the tnecal reaction of atretic antral foll icles (a) •

>:36

J.

" . ,

,

..

, •

•

,

• • •

(j)

c

-.r. l ~

1

• ~ .. ;

",'-.) \

.0", .

FIGURE 9

FIGURE 10

FIGURE Il

FIGURE 12

Light micrograph showing a secondary interstitial cell cluster from a control ovary imrnunostained for AP (area dernarcated by arrows). These cells show a weak reaction to the imrnunosta in 1 in contra st to the reaction of foll icular thccal cells (large arrowheads) and vascular endothelial cells (srnall arrowheads).

x468

Light rnicrograph of AP-positive secondary intersti tial cell strands (arrowheads) and a secondary intersti tial cell cl uster (arrow) from a rnicrocystic ovary. The AP posi ti ve structure at the tc:p of the field represents thecal cells encircling a primary follicle.

x360

Light micrograph from a macrocystic ovary immunostained for AP. The highly reactive thecal cell layer on the bottom (h) belongs to a healthy secondary follicle, in contrast to the moderately reactive one on the top which encircles a precystic follicle (pc).

x337.5

Light rnicrograph of the theca l cell laye: r of a heal thy secondary foll icle (h) and of a microcyst (c), immunostained for AP. The reaction in the microcystic thecal layer is primarily over endothelial cells, whereas bath thcca land endothelial cells (arrowheads) in a sccondary follicle are AP positive.

x540

C ~® ~

1 1

1

~.;, \ ,

", . ~ .... ~, . ,

r •

t ... ,/ , , ~ " Il'

~. " '. \. '" , '- .-

pc ® @

4c • t~ •

.., ... ~ -

" - ---, .~ ........ or 4

.,-~ -"-{~ .. ,

..... ,- c ,~ h '1. ... (

,.- .. "

1

FIGURE 13 Mean Cell Size of Thecai and Secondary Intersti tial Celis in an EV-Treated (Macrocystic) Ovary

This histograrn represents the rnean size of cells (n=20) from rnacrocystic avaries + standard error of the mean (S. E .M. ). Heal thy secondary thccai cells are significantiy smaller than the other four cell types presented (p<.OOl). Macrocystic thecal cells are significantly iarger than precystic follicuiar thecal cells (p<. 05) .

Mean Cell Size Macrocystic Ovary

16~----------------------------------------------------------------------~

14

ht'nllhy 2 alrellc 2" precysllc cystlc 2" mlerstillai

thocal cell type

FIGURE 14

FIGURE 15

Electron micrograph of the membrana granulosa of a precystic follicle in a macrocystic ovary. Note the variability in cell and nuclear shape. cytoplasmic blebbing of the apical surfaces is prominent on those cells in the right side of the field. Lipid droplets (L) and autophagie vacuoles (a), characteristic of a follicle in an advanced state of atresia, are labelled for reference. Luminal surface is at the top of the field.

x3,000

Electron micrograph of the membrana granulosa of a maerocystic follicle. It eonsists of a single layer of cells characterized by highly indented nuclei and large, irregular intercellular spaces. Luminal surface is at the top of the field.

x3,500

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FIGURE 16 Electron microscopie radioautograph of thecal cells from a heal thy secondary follicle. Grains represent binding of 125I _hCG to LH/hCG receptors. Lipid appears as empty spheroidal inclusions.

x13,500

'.

1

•

1

FIGURE 17 Electron microscopie radioautograph of a thecal cell from an atretic secondary folliele, photographed at the same magnifieation as Figure 16. In general, this cell is larger and more irregular in shape than healthy secondary follicular thecal cells. Its ultrastructural characteristics are similar.

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FIGURE 18a Electron microscopie radioautograph of a theeal cell from a preeystic follicle (Ev-treated ovary). The lipid droplets in these eells are significantly smaller than those within atretic and cystic theeal cells. Note the euchromatic state of the nucleus.

x12,600

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FIGURE 18b Electron microscopie radioautograph of a thecal cell from a macrocystic follicle. These cells are significantly larger than precystic thecal cells. These cells conta in the largest l ipid droplets. This cell was located near the basal lamina of the cystic follicle and is, consequently a srnaller representati ve rnacrocyst ic thecal celle Note the heterochrornatic state of the nucleus.

x12 , 600

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FIGURE 19 Electron microscopie radioautograph of a secondary interstitial cell from a maerocystic ovary. These polygonally shaped cells are very large and give off numerous cytoplasmic projections. They contain an abundanee of lipid droplets of a wide variety of sizes, and mitochondria with tubulovesicular cristae. Note the euchrornatie state of the nucleus.

x12,600

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FIGURE 20 Mean Area of Lipid Droplets in Thecal and Secondary Interstitial Cells in a Macrocystic Ovary

This histogram represents the rnean area of lipid droplets ± S. E. M. The mean area of l ipid droplets within thecal cells of atretic secondary follicles is significantly larger than the mean lipid droplet area of he;;:llthy and precystic follicular thecal c81ls and sccondary intersti tial cells (p<. 002) . Lipid droplets within macrocystic thecal cells are significantly larger than those within the other four classes of cells studied (p<. 05) . There are no significant differences in the percentage of cel1 profile area occupied by lipid in the sc five different cell types. (n=10 cells/typc).

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-

FIGURE 21 Mean Area of Mitochondriai Profiles in Thecal and Secondary Interstitiai Cells in a Macrocystic Ovary

This histograrn represents the rnean area of mitochondriai profiles ± S.E.M. Mitochondriai profiles within atretic secondary follicular thecal celis are significantly srnailer than those within thecal cells of healthy secondary, precystic and cystic follicular origin (p<.05). As weIl, the percentage of the atretic thecal cell profile area occupied by mitochondriai profiles is also significantly srnaller than in heal thy secondary and precystic thecal cells and secondary interstitial cells. Other differences are not significant. (n=lO cells/type).

4

3

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Mean Mitochondrial Area Macrocystic Ovary

1 " " ... ,- ,- ,-

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aIrelle 2' precystlc cySllc

lhecal cel! type

... " .- .-" "

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, ... " ,- " " ,- ,-... ... ... ... , , ... ... ... .- .- ... .- .- " ... .- .- ,-... ... ... ... , ... ... ... ... .- .- .- ./' ... ,- ...... ... ,. 2° Inlerslillui

FIGURE 22 Analysis of LH/hCG Binding sites on Thecal and Secondary Intersti tial Cells in a Macrocystic Ovary Using Electron Microscopie Radioautography

Summary of the nurnber of 125 I _hCG positive si tes/l, OOOIrn surface length on elcetron microscopie radioautographs. Di fferences in the mean nurnber of grains/1000im between healthy and atretie secondary follicular theeal cells are not signifieant. Pl eeystic thecal cells possess signifieantly fewer binding si tes than heal thy and atretic follieular thecal eells (p<. 005) . cystic thecal cells possess significantly fewcr binding sites than aIl five cell types (p<.OOl). The rnean nurnber of grains associated with seeondary intersti tial eells is significantly greater than the number associated with atretic, precystic and cystic theeal cells (p<.006), but is not significantly different than the nurnber of sites assoeiated with thecal eells of hcal thy secondary follicles. (n=10 cells/type).

S g> .!!! Il> <> ~ ::1 II)

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Number of Grains per 1,OOOum Surface Length Macrocystic Ovary

20.-------------------------------~------~-------------------------;

15

1

10

5

IWillthy 2" dtrctlC 2" precystlc cystlc

thecal cell type

FIGURE 23 Mean Cell Size of Thecal and Secondary Interstitial Celis in Ez-Implanted (Microcystic) ovaries

This histogram represents the mean size of cells (n=20) from microcystic ovaries ± S.E.M •• Atretic secondary follicular thecai celIs are signific...:antIy larger than those of a heal thy secondary follicie (p<.OOl). Secondary interstitiai celi size is significantly larger than healthy and atretic follicular thecal celi size (p<.05).

FIGURE 24 Electron micrograph of the membrana granulosa of a microcystic follicle, which possesses characteristics of the membrana granulosa of both precystic and cystic follicles in an Ev-treated (macrocystic) polycystic ovary. Cells are irregularly shaped, give off numerous cytoplasmic extensions, and are held together by only a few remnant junctional complexes. Autophagie vacuoles are occasionally seen (upper right of field), while lipid droplets are not. A segment of basement membrane appears in the lower left corner of the field.

X5,300

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FIGURE 25 Electron microscopie radioautograph of a thecal cell from a microcystie follicle. These cells are approximately the same size as atretic secondary follicular thecal eells. The size of lipid droplets in these cells is significantly larger than heal thy and atret ic foll icular thecal and secondary intersti tial cell l ipld droplets. Like macrocystic thecal cells, these cells possess very few LH binding sites. Note the heterochromatic st?ce of the nucleus.

x12,600

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FIGURE 26 Electron microscopie radioautograph of a seeondary interstitial cell from a rnicrocystie o\l'~ry. This cell is very similar morphologically to i i.:s eounterpart in a macrocystic ovary, containing an abundanee of lipid droplets of a wide variety of sizes, and rnitoehondria with tubulovesicular cr istae. These cells contain significantly more LH/hCr; binding sites than any other thecal cell txpe in il microcystic ovary, and appear to bind 1 I-hCG more extensively than macrocystie secondary intersti tial cells. Note the euchromatic state of the nucleus.

x12 , 600

•

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FIGURE 27 Mean Area of Lipid Droplets in Thecal and Secondary Interstitial Cells from a Microcystic Ovary

This histogram represents the mean area of l ipid droplets ± S. E .M. There are no significant differences between Mean area of lipid droplets within healthy and atretic secondary follicular or secondary intersti tial cells. The Mean 1 ipid droplet area of cystic thecal cells is significantly larger than the other three cell types under study in these ovar ies (p<. 007) . (n=10 cellsjtype).

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FIGURE 28 Mean Area of Mitochondrial Profiles in Thecal and Secondary Intersti tial Cells in Microcystic Ovaries

This histogram represents the mean area of mitochondrial profiles ± S.E.M. The mean are a of mitochonctrial profiles within secondary interstitial cells is significantly Iarger than rnitochondrial profiles of atretic and cystic follicular origin (p<.04). There are no significant differences between the proportion of the total cell profile area occupied by rnitochondrial profiles in these four cell types, howeve~. (n=10 cells/type).

FIGURE 29 Analysis of LH/hCG Binding sites on Thecal and Secondary Interstitial Cells in a Microcystic Ovary Using Electron Microscopie Radioautography

Summary of the number of 125I _ hCG binding sites/1,OOOIm surface length on electron microscopie radioautographs. Differences in the mean number of grains/1000Im between heal thy and atretic secondary follicular thecal cells are not significant. Microcystic thecal cells possess significantly fewer LH binding si tes than the other three cell types examined in these ovaries (p<. 001) . Secondary intersti tial cells have a significantly larger number of LH receptors as compared to atretic secondary and microcystic thecal cells (p<. 004). (n=10 cells/type).

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