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
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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!
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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.
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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.
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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
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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
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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.
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(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 ,,/
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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.
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RO
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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
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1
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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.
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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).
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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).
t
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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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