A Study of the Cumulus-Oocyte Complex (2006)

8/13/2019 A Study of the Cumulus-Oocyte Complex (2006)

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CELL-TO-CELL COMMUNICATION AND OVULATIONA Study of the Cumulus-Oocyte Complex

N O R T O N B . G I L U L A , M I L E S L . E P S T E I N , an d W I L L I A M H . B E E R SFrom The Rockefeller University, New Y ork 10021. Dr. Epstein's present address is the Depa rtmentof A natomy, University of W isconsin School of Medicine, M adison, Wisconsin 53706.

A B S T R A C TC e l l - to - c e l l c o m m u n i c a t i o n w a s c h a r a c t e r i z e d i n c u m u l u s - o o c y t e c o m p l e x e s f r o mr a t o v a r i a n f o l li c le s b e f o r e a n d a f t e r o v u l a t i o n . N u m e r o u s , s m a l l g a p ju n c t i o n a lc o n t a c t s w e r e p r e s e n t b e t w e e n c u m u l u s c e l l s a n d o o c y t e s b e f o r e o v u l a t i o n . T h eg a p j u n c t i o n s a r e f o r m e d o n t h e o o c y t e s u r f a c e b y c u m u l u s c e ll p r o c e s s e s t h a tt r a v e r s e t h e z o n a p e l l u c i d a a n d c o n t a c t t h e o o l e m m a . T h e e n t i r e c u m u l u s m a s sw a s a l s o c o n n e c t e d b y g a p j u n c ti o n s v i a c u m u l u s - c u m u l u s i n t e r a c t i o n s . I n t h eh o u r s p r e c e d i n g o v u l a t i o n , t h e f r e q u e n c y o f g a p j u n c ti o n a l c o n t a c t s b e t w e e nc u m u l u s c e ll s a n d t h e o o c y t e w a s r e d u c e d , a n d t h e c u m u l u s w a s d i s o r g a n iz e d .E l e c t r o p h y s i o l o g i c a l m e a s u r e m e n t s i n d i c a te d t h a t b i d i r e c t i o n a l i on i c c o u p l i n g w a sp r e s e n t b e t w e e n t h e c u m u l u s a n d o o c y t e b e f o r e o v u l a t i o n . I n a d d i t i o n , i o n t o p h o -r e t ic a l ly i n je c t e d f l u o r e s c e i n d y e w a s t r a n f e r r r e d b e t w e e n t h e o o c y t e a n d c u m u l u sc e ll s . E x a m i n a t i o n o f th e e x t e n t o f i o ni c c o u p l i n g in c u m u l u s - o o c y t e s p e c i m e n sb e f o r e a n d a f t e r o v u l a t i o n r e v e a l e d t h a t i o n i c c o u p l i n g b e t w e e n t h e c u m u l u s a n do o c y t e p r o g r e s s i v e l y d e c r e a s e d a s t h e t i m e o f o v u l a t i o n a p p r o a c h e d . I n p o s t o v u -l a t o r y s p e c i m e n s , n o c o u p l i n g w a s d e t e c t e d . A l t h o u g h s o m e p r o t e o l y t i c m e c h a -n i s m m a y b e i n v o l v e d in t h e d i s i n te g r a t i o n o f t h e c u m u l u s - o o c y t e c o m p l e x ,n e i t h e r t h e c u m u l u s c e ll s n o r t h e o o c y t e p r o d u c e d d e t e c t a b l e l e v e l s o f p l a s m i n o -g e n a c t i v a t o r , a p r o t e a s e w h i c h is s y n t h e s i z e d b y m e m b r a n a g r a n u l o s a c e ll s.

I n s u m m a r y , c e l l c o m m u n i c a t i o n i s a c h a r a c t e r is t i c f e a t u r e o f t h e c u m u l u s -o o c y t e c o m p l e x , a n d t h is c o m m u n i c a t i o n i s t e r m i n a t e d n e a r t h e t i m e o f o v u la t i o n .T h i s te m p o r a l p a t t e r n o f th e t e r m i n a t i o n o f c o m m u n i c a t i o n b e t w e e n t h e c u m u l u sa n d t h e o o c y t e m a y i n d i c a t e t h a t c o m m u n i c a t i o n p r o v i d e s a m e c h a n i s m f o rr e g u l a t i n g th e m a t u r a t i o n o f t h e o o c y t e d u r i n g f o ll i c ul a r d e v e l o p m e n t b e f o r eo v u l a t i o n .

K E Y W O R D S c e l l communication gapjunctions - ion ic electrotonic) coupling dyetransf er cumulus-oocy te om plex ovu lat ionT h e av ascu l a r co m p ar tm en t o f t h e m am m al i anovar ian fo l lic le con tains several d i f feren t types o fcel ls whose metabo l ism and funct ion must beprecisely con tro l led dur ing the hours p reced ing

o v u la t i o n . S in ce a n u m b er o f k n o w n h o rm o n e-d ep en d en t m e tab o l i c an d m o rp h o lo g ic ch an g esoccur dur ing th is ra ther b r ief t ime per iod (4 ) , thefo l l ic le rep resen ts a un ique system to s tudy theregu lat ion o f hetero logo us cel l in teract ions dur ingdevelopment and d i f feren t ia t ion .The ma jo r popu la t ion o f cel ls wi thin the fo l lic-u lar an t rum are ep i thel ia l g ranu losa cel ls. W ith in

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t h i s p o p u l a t i o n , t h e r e i s a d e f i n i t e h e t e r o g e n e i t yw i t h r e s p e ct t o m o r p h o l o g y a n d h o r m o n e b i n d i n g .F o r e x a m p l e , r e c e p to r s f o r h u m a n c h o r i o n ic g o-n a d o t r o p i n a r e p r e s e n t o n g r a n u l o s a c e l l s i n t h ep e r i p h e r a l r e g i o n o f t h e f o l li c le w h e r e a s t h e g r a n -u l o s a c e l l s n e a r t h e a n t r a l c a v i t y a n d t h e o o c y t eh a v e f e w d e t e c t a b l e r e c e p t o r s f o r t h e h o r m o n e( 1 ) . I n a d d i t i o n , s o m e o f t h e g r a n u l o s a c e l l s a r ep h y s i c a l l y a s s o c i a t e d w i t h t h e f o l l ic l e w a l l , s o m ew i t h t h e o o c y t e , s o m e w i t h o t h e r g r a n u l o s a c e l ls ,a n d s o m e w i t h t h e a n t r a l f l u id .

T h e g r a n u l o s a ce ll s t h a t s u r r o u n d t h e m a m m a -l ia n o o c yt e a r e k n o w n a s t he c u m u l u s o o p h o r u s .T h e i n n e r m o s t l a y e r o f c e l ls in t h e c u m u l u s , t h ec o r o n a r a d i a t a , s e n d s c y t o p l a s m i c p r o c e s s e st h r o u g h t h e i n t e r v e n i n g z o n a p e l l u c i d a t o c o n t a c tt h e o o l e m m a ( 9 , 2 0 , 2 6 , 3 1 ) . T h e r e h a v e b e e ns e v e r a l re p o r t s t h a t g a p j u n c t i o n s a r e p r e s e n t i nt h e r e g i o n s o f c o n t a c t b e t w e e n t h e s e h e t e r o l o g o u sc e l ls ( c u m u l u s o o c y t e ) ( 2 , 3 , 2 8 ) . S i n c e g a p ju n c -t i o n s h a v e b e e n i m p l i c a t e d a s a s t ru c t u r a l p a t h w a yf o r c e l l - to - c e l l c o m m u n i c a t i o n ( 7 , 1 6 , 1 7 , 2 3 ) , 1 i th a s b e e n t e m p t i n g t o s p e c u l a t e th a t t h e p r e s e n c eo f t h e s e j u n c t i o n a l e l e m e n t s m a y i n d i c a t e t h a tt h e r e i s a s i g n i f ic a n t e x c h a n g e o f m o l e c u l e s b e -t w e e n t h e c u m u l u s c e ll s a n d o o c y t e d u r i n g f o l li c u -l a r d e v e l o p m e n t . T h i s p o s s i b i li t y m a y b e o f c o n -s i d e r a b l e i m p o r t a n c e i n t e r m s o f o o c y t e m e t a b o -l i s m a n d m a t u r a t i o n s i n c e i t h a s b e e n s u g g e s t e dt h a t c u m u l u s c e ll s a r e r e s p o n s i b l e f o r p r o v i d i n gs e v e r a l t r o p h i c o r m e t a b o l i c f a c t o r s t o t h e p r e o -v u l a t o r y o o c y t e ( 8 , 1 2 , 1 5 , 2 2 ) . R e c e n t l y , i t h a sb e e n d e m o n s t r a t e d t h a t [ a H ] u r i d i n e c a n b e in c o r -p o r a t e d i n t o o o c y te R N A i n i n t ac t m o u s e c u m u -l u s - o o c y t e p r e p a r a t i o n s , w h e r e a s i t is n o t i n c o r -p o r a t e d i n id e n t i c a l p r e p a r a t i o n s w h e r e t h e c u -m u l u s h a s b e e n r e m o v e d ( 3 0 ). A s i m i la r c u m u l u sc e l l d e p e n d e n c y f o r g r o w t h o f m o u s e o o c y t e s h a sa l s o b e e n r e p o r t e d ( 1 3 ) .

B e f o r e t h e t i m e o f o v u l a t i o n , t he n u m b e r o fc u m u l u s c e l l p r o c e s s e s t o t h e o o c y t e d e c r e a s e s( 1 9 , 2 0 , 2 6 ) , a n d t h e r e i s a h o r m o n a l l y i n d u c e de l e v a t e d c o n c e n t r a t i o n o f p l a s m i n o g e n a c t i v a t o ri n t h e f o l l i c u la r f lu i d t h a t i s p r o d u c e d b y t h eg r a n u l o s a c e l l s ( 6, 2 7 ) . A f t e r o v u l a t i o n f e w , i fa n y , c u m u l u s - o o c y t e p ro c e s s e s a r e e v i d e n t . T h u s ,e v e n t u a l l y , t h e o o c y t e l o s e s a l l p h y s i c a l c o n t a c tw i t h i t s a s s o c i a t e d c u m u l u s c e l l s .T h e e x p e r i m e n t s d e s c r i b e d i n t h i s p a p e r w e r e1 In this pape r , the term s cel l- to-cel l comm unica t ion andin t e r ce ll u l a r comm unica t ion a r e used to r e f e r t o t heprocess of t ransfer of cytoplasmic substances betweencel ls via a low-resistance junc t ional mecha nism .

u n d e r t a k e n i n o r d e r to d e t e r m i n e t h e s t r u ct u r a la n d p h y s i o l o g ic a l b a s i s f o r c e l l -t o - c e ll c o m m u n i -c a t io n i n t h e r a t c u m u l u s o o p h o r u s - o o c y t e c o m -p l e x . I n a d d i t i o n , t e m p o r a l c h a r a c t e r i s ti c s o f c o m -m u n i c a t i o n h a v e b e e n e x a m i n e d d u r i n g t h e s ta g e so f fo l l i c u la r d e v e l o p m e n t t h a t l e a d t o t h e o v u l a -t o r y e v e n t . A p r e l i m i n a r y r e p o r t o f t h e s e s t u d i e sh a s b e e n p r e s e n t e d p r e v i o u s l y ( 1 4 ) .M A T E R I A L S A N D M E T H O D SMaterials

Imm ature female Sprague-Dawley rats (26 days old)wer e ob t a ined f r om Hol t zman Co . ( Mad i son , Wis . ) .Other mater ials ut i l ized in this study include: pregnantmar e se r um gonado t r op in ( PMSG) ; human chor ion i cgonadotropin (HCG); poly-L- lysine, mol wt 70,000( S igma Chemica l Co . , S t . L ou i s , Mo . ) ; med ium 199 ,and penici l l in-st reptomy cin (Gra nd Is land BiologicalCo . , G r and I s l and , N . Y . ) ; f et a l bov ine se r um ( Rehe i sChemical C o., Chicago, I11.); Falcon cu l ture dishes(BioQuest , BBL & Falcon Products, Cockeysvi l le ,Md.) ; disodium f luorescein and osmium tet roxide (4aqueous) (Polysciences, Inc. , Warr ington, Pa.) ; g lutar -aldehyde solut ion (50 wt/wt , biological grade ) , para-formaldeh yde, and u rany l acetate (Fish er Scient if ic Co. ,Pi t tsburgh, Pa.) ; Ep on 812; tannic acid, analy t ical grade(MaUinckrodt , Inc. , S t . Louis, Mo.) ; and omega dottub ing ( Hi lgenberg Glass , Ger m any) .Preparation of Cumulus-Oocyte Complexesand Granulosa Cells

Fem ale Sprague-Dawley rats, 26 days old, were in-j ec t ed subcu taneous ly wi th 5 I U o f PMS G in 0 .1 ml o f0 .9 NaCI . Af t e r 48 h , t he an imal s wer e admin i s t e r ed25 IU of HCG. With this regimen, f ive to six fol l iclesper ovar y wi ll ovu la t e 10 - 12 h a f t e r t he H CG in j ec t i on .Preovulatory fol l icles are dist inguishable f rom othergraaf ian fol l icles in the ovary on the basis of size andco lo r a t ion appr ox . 4 h a f t e r the H CG in j ec t i on .Cum ul i and g r anu losa cel ls wer e p r epar ed f r om ind i -vidual fol l icles at var ious stages of development by thefol lowing procedure. The rats were decapi tated and theovar ies were dissected f ree of adher ing t issue in thep r esence o f med ium 199 wi th an t ib io t i c s ( 100 U /mlpenici l l in and 100 ~.g/m l st reptom ycin) . Fol l icles werepunc tu r ed wi th a 26 - g need le a nd the i r con ten t s ex -p r essed in to t he med ium. T he p r epar a t i on was t henexamined a t 1 0 - 20 x magn i f i ca t ion , and cumu l i wer er emoved w i th a f i ne p ipe t te . T hese w er e t r ansf e r r ed t of resh m ed ium, washed by r epea t ed t r ansf e r s and thendeposi ted in a t issue cul ture vessel con taining medium199. The rema ining granulosa cells were then col lectedby cen t r i f uga t ion , washed a nd p l a t ed a t t he des i r eddensi ty in [n~I] f ibr in-coated cul ture vessels (27) andanalyzed for plas mino gen act ivator as de scr ibed below.

Postovulatory cum uli were isolated f rom oviducts 2 2-

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29 h after the HCG injection (10-17 h after ovulation)by the following method. The ovary and oviduct weresurgically removed from the animal and placed in me-dium 199. The oviduct was then excised from the ovaryand placed in fresh medium. In the e xtended oviduct, abolus of 5-6 cumuli could be located and visualized withthe aid of a dissecting microscope at 10-20 x. Once thecumuli were located, the oviduct was cut immediatelydistal to the bolus, and the bolus was then expelled intothe me dium by' oviductal peristalsis. The postovula torycumuli were then recovered from the culture dish andutilized for the physiological or biochemical determina-tions.Ligh t Microscopy

Light mierographs of follicles from appropriatelytreated animals were obtained by cutting 1 /zm thicksections of Epon-embedded specimens that were fixedand processed for electron microscopy. The 1 /~m thicksections were mounted on glass slides and stained withtoluidine blue. All photomicrographs were taken withbright field optics in a Zeiss Photomicroscope II.

lectron MicroscopyTh in SECTIONS: Ovarian samples from appro-priately pr imed animals were fixed with a combina tion

of 4% glutaraldehyde (vol/vol) and 0.5% paraformal-dehyde (wt/vol) in 0.1 M cacodylate buffer (pH 7.3) for2 h at room temperatur e. At this time, the follicles weredissected as intact tissue fragments for subsequent proc-essing. Care was taken to avoid rupturing the follicles.Regions of oviduct that contained a bolus of postovula-tory material were selected with a dissection microscopeand processed as intact tissue segments. After initialaldehyde fixation, the samples were treated for 1 h with1% (vol/vol) osmium tetroxide in veronal-acetate buffer,followed by buffer rinses. Then the samples were treate den bloc with 1% tannic acid in 0.05 M sodium caeodylatebuffer (pH 7.0) for 45 min, washed with 1% sodiumsulfate in 0.1 M sodium cacodylate buffer (pH 7.3) for10 min, and then treated with uranyl acetate in veronal-acetate buffer for 1 h. The samples were dehydrated ina graded series of ethanol (70%, 95%, 100%), andembedded in Epon 812. Thin sections were cut with adiamond knife on a Sorvall Porter-Blum MT2-B Ultra-microtome and mounted on copper grids. The sectionswere stained with uranyl acetate and lead citrate beforeexamination in a Philips 300 transmission electron mi-croscope at 80 kV.

FREEZE-FRACTURe: Approp riat e follicles weredissected from intact ovaries that had been fixed for 2 hat room tempera ture in a mixture of 4% glutaraldehydeand 0.5 % paraformaldehyde in 0.1 M sodium cacodylatebuffer (pH 7.3). The samples were then treated for 2- 4h at room temperature with 25% glycerol in 0.1 Mcacodylate buffer before rapid freezing in Freon 22 andstorage in liquid nitrogen. The samples were freeze-

fractured and replicated with a carbon-platinum mixturein a Balzers BM 360 apparatus at -115~ The replicaswere cleaned in bleach and distilled water before mount-ing on 300-400-mesh grids. All electron microscopeobservations were made with a Philips 300. The freeze-fracture images have been mounted so that the shadowdirection is from the bottom to the top of the micro-graphs.Physiology

Microelectrode impal ements were significantly facili-tated by attaching the cumulus-oophorus complexes toculture dishes that had been pretreated with poly-L-lysine. Optimal attachment results were obtained bytreating the culture dishes overnight with 0.1% (wt/vol)poly-L-lysine. This treatment was followed by rinsingwith distilled water, and then adding medium 199 beforethe addition of the biological samples.

ELECTROPHYSIOLOGICAL RECORDING: Dishescontaining cumuli were placed on the stage of an in-verted microscope (Leitz Diavert) and recording wascarried out at room temperat ure. Preparations were usedfor a maximum of 3 h after the rat was killed. Microelec-trodes, made from omega dot tubing, were bent on anicrome wire to approx. 45 ~ and filled with 2.8 M KClto give resistances of 50-70 MfL The microelectrodeswere connected through Ag/AgCl wires to the input ofcapaci ty-compensated DC amplifiers. One microelec-trode served to record membrane potential and the otherelectrode was used to inject pulses of current through abridge circuit. The indifferent electrode was a 0.5-mmcapillary tube filled with Ca,Mg-free PBS dissolved in 2-3% agar. This tube connected a medium-filled reserviorcontaining a Ag/AgCl lead with the culture dish. Thehath was held at virtual ground by connecting theindifferent electrode to the summing junction of a Phil-brick P24 operational amplifier (Teledyne Philbrick,Dedham, Mass.) used to monitor current intensity.Current pulses for stimulation were provided by Tektro-nix pulse generat ors (Tektronix, Inc., Beaver ton, Ore.) .Membrane potentials and current intensity were dis-played on a Tektronix 5111 storage oscilloscope andphotographed with a kymograph camera. The image ofthe cells to be penetrated was projected through themicroscope and photographed on 35 mm film. A mini-mum resting membrane potential of - 10 mV was re-quired before a cell was used for the coupling studied.

Y E INJECTIONS: For the dye trans fer experi-ments, electrodes (approx. 10 Mft when filled with 2.8M KCI) were filled with 5% (wt/vol) disodium fluores-cein, dissolved in distilled wate r or 0.1 M KCI. Negativecurrent pulses (7-10 10 -9 A) of approx. 0.5 s durationwere injected at a frequency of 1/s. Fluorescein wasvisualized through bright-field optics using a 100-Wtungsten halogen lamp, a combination of BG 12, 23,and 38 excitation filters, a KP 490 cut-off filter and a KP530 barrier filter, and re corded on Tri-X film processedin Aeufine.

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Plasminogen Activator DeterminationsCumuli or granulosa cells were analyzed for plasmin-ogen activator by a previously described method (27).The cells or cumuli were plated in medium 199 contain-ing 10% plasminogen-depleted fetal bovine serum on a

layer of [mI]fibrin which had been previously formed onthe bottom of the culture vessel. After 12 h, the mediumwas removed, and after the cultures were washed twicewith medium 199, fresh medium 199 containing 10%acid-treated fetal bovine serum was added. Aliquots ofthe medium were then removed and analyzed for soluble[lzsI]fibfin peptides in a Packard autogammacounter(Packard Instrument Co., Inc. , Downers Grove, I11.) atdesired times.Plasminogen-depleted (21) and acid-treated (low pro-tease inhibitor) (18) fetal bovine sera were prepared aspreviously described.Plasminogen was recovered from the lysine-sepharosecolumns used to plasminogen-deplete the serum by themethod of Deutsch and Mertz (11). Conversion of thiszymogen to plasmin by urokinase was achieved as previ-ously described (5).

RESULTSThere are a number of hormonally controlledstages in follicular developmen t that are associatedwith major cytological changes in the cumulus-oocyte complex. Acute a lterations in the complex,as well as in the maturation stages of the oocyte,take place in preovulatory follicles just beforefollicular rupture at ovulation. In this study, spe-cific stages of follicular development were exam-ined in order to d etermin e (a) whether cell-to-cellcommunicat ion exists within the oocyte complex,and (b) whether there are any changes in com-munication that might be related to the events ofoocyte maturation and ovulation.Light Microscopy

The following stages of cumulus-oocyte devel-opment were examined in this study: (a) Speci-mens were prepared from rats 48 h after PMSGinjection. At this time, a large number of antralfollicles are present in the ovary, but ovulation hasnot occurred. T he cumulus-oocyte complex in oneof these follicles is shown in Fig. 1 A. (b) Ratswere administered an ovulatory regimen of PMSGand HCG. Shortly before ovulation, two classesof large follicles are evident. The first, thosedestined to ovulate, can be distinguished on thebasis of their pink color and the fact that theyprotrude well above the surface of the ovary. Thecumulus-oocyte complex from one of these preo-

vulatory follicles is shown in Fig. 1 C. T he secon d,and by far the larger class, consists of large antralfollicles that do no t ovula te. Fig. 1 B is a micro-graph of a cumulus-oocyte complex from such afollicle, which was prepa red from the same ovaryat the identical time as the preovulatory follicle inFig. 1 C. (c) Finally, postovulatory samples wereobtained from the oviduct approximately 10 hafter ovulation (Fig. 1 D).

In antral follicles that are not preovulatory (Fig.1A and B), the cumulus-oocyte complex is char-acterized by a compact region of stratified cuboi-dal epithelial cells (cumulus oophorus) that areseparated from the oocyte by a clear region, thezona pellucida. The striations in the zona pellucidaare due to the cytoplasmic processes that connectthe cells of the innermost cumulus layer, thecorona radiata, with the oocyte.

In late preovulatory follicles (Fig. 1 C) dramaticchanges are evident in the complex. The cumulusis less compact, and it is frequently reduced to asingle layer of cells, the corona radiata. Thecumulus mass is essentially reduced to a popula-tion of dissociated cells. The corona radiata is in-terrupted by regions of missing cells, and thecumulus cells exhibit short thick cytoplasmic proc-esses or blebs. In addition, there are few, if any,detectable striations across the zona pellucida. Bycomparing the appearance of complexes in folli-cles that are not preovulatory, either before (Fig.1 B) or after (Fig. 1A ) H CG administra tion, withthe late preovulatory sample (Fig. 1 C), it wouldappear that the structural disintegration of thecumulus is a direct correlate of ovulation, and no ta general response to elevated gonadotropin lev-els.

The postovulatory oocyte is still surrounded bya zona pellucida and cells from the cumulus.However, the structural disintegration of the cu-mulus oophorus is even more pronounced at thistime than it was near the time of ovulation. Somecells of the corona radiata remain attached to thezona; however, most of the dissociated cells areretained in the vicinity of the oocyte because ofthe extracellular matrix that is responsible forgenerating a bolus of oocytes in the oviduct.

Electron MicroscopyThe cumulus-oocyte complex in follicles that

are not preovulatory is characterized by a largenumber of cytoplasmic prrocesses that can bedetected in the zona pellucida (Fig. 2). These

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Fi6 tr~ 1 Light micrographs of the cumulus-oocyte complex a t var ious s tages of developm ent. A)Appearance of a complex in a fol l ic le f rom a ra t tha t had been trea ted with PMSG 48 h ear l ie r . Themult i layered cumulus cel l region is separa ted from the oocyte by the zona pel luc ida . B) Complex from anonpreo vula tory foll ic le . Th e experimen ta l animal had been adminis tered an ovula tory regimen of PMSGand HC G. This sample was taken 10 h af ter HC G injec t ion, which is near the t ime tha t ovula t ion can beexpected to occur . Nonpreovula tory samples , such as this one , were examined routine ly as internalcontrols s ince the ir morphologica l and physiologica l propert ies c lose ly resembled the ones from largefoll ic les found in PMS G-trea ted animals A). C) Com plex from a preovula tory fol licle prepared from thesame ovary as tha t described in 8) . The specimen was designated preovula tory on the basis of i ts s ize andcolor . The cumulus is reduced to a s ingle layer of ce l ls tha t appear to be separa ted from each other insome regions . D) Postovula tory specimen obta ined from the oviduct about 10 h af ter ovula t ion. Somecumulu s cells are still loosely associated with th e occyte at thi s time. A-D) x 420.

p r o c e s s e s a r e e x t e n s i o n s o f t h e e p i t h e l i a l c u m u l u sc e l l s . T h e y t r a v e r s e t h e z o n a p e l l u c i d a a n d c o n t a c tt h e s u r fa c e m e m b r a n e o f t h e o o c y t e , t h e o o -l e m m a . A t l o w m a g n i f i c a t i o n , t h e c u m u l u s c e l lp r o c e s s e s , w h i c h r e s e m b l e p r e s y n a p t i c b o u t o n s ,c a n b e r e a d i l y d i s t i n g u i s h e d a t t h e o o c y t e s u r f a c es i n c e t h e y a r e m o r e d e n s e t h a n t h e o o c y t e c y t o -p l a s m . T h e r e g i o n s o f h e t e r o l o g o u s c e l l i n t e r a c t i o na p p e a r t o b e r a n d o m l y d i s t r i b u t e d o v e r t h e e n t i r es u r f a c e o f t h e o o c y t e , a n d t h e o o c y t e m i c r o v i l l ia r e d i s p l a c e d t o a c c o m m o d a t e t h e c o n t a c t i n gproc e s se s F igs . 2 a nd 8) .

A t t h e s i te s o f i n t e r a c t io n b e t w e e n t h e c u m u l u s

p r o c e s s e s a n d t h e o o l e m m a , t w o p r o m i n e n t t y p e so f c e l l c o n t a c t a r e p r e s e n t : O n e o f t h e s e i s ad e s m o s o m e , t h e o t h e r i s a g a p j u n c t i o n . I n t h i ns e c t i o n s , t h e d e s m o s o m e w h i c h c l o s e l y r e s e m b l e sa f a sc i a a d h a e r e n s i s f r e q u e n t l y s e e n a s a b i p a r t i t es t r u c t u r e c o m p o s e d o f o o c y t e a n d c u m u l u s c e llp r o c e s s e l e m e n t s F i g s . 3 a n d 4 ) . I t s p r e s e n c e i si n d i c a t e d b y a n e l e c t r o n d e n s i t y o n t h e c y t o p l a s -m i c su r f a ce s o f t h e m e m b r a n e s , a n d t h e a b s e n c eo f a n e l e c t r o n - d e n s e m a t r i x i n t h e i n t e r v e n i n gi n t e r c e l l u l a r s p ac e . A l t h o u g h t h e r e i s n o d i s t i n c ti n t e r n a l m e m b r a n e d i f f e r en t i a t i o n t h a t i s c h ar a c -t e r i st i c o f t h e s e d e s m o s o m e s , a l o o s e a g g r e g a t e o f

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F IG URE 2 T h i n s e c t i on o f t he c um ul u s - ooc y t e c om p l e x i n a la r ge nonpr e o vu l a t o r y f o l li c l e f r om a r a tt r e a t e d w i t h P M S G a n d H C G . T h e s p e c i m e n w a s t a k e n 1 0 h a f t e r th e H C G i n j e c ti o n . C e ll s o f t h ei n n e r m o s t c u m u l u s l a y e r , t h e c o r o n a r a d i a ta , s e n d p r o c e s s e s t h r o u g h t h e z o n a p e U u c id a ( Z P ) t o f o r mc on t a c t s on t h e su r f a c e o f t he ooc y t e ( sm a l l a r r ow s ) . I n t h i s i m a ge , t h e c um ul us p r oc e s se s a r e e a s i lyd i s t i ngu i she d f r om t he shor t m i c r ov i l l i on t he ooc y t e su r f a c e by t he d i f f e r e nc e i n s t a i n i ng de ns i t y . T hec um ul us c e l l s a r e a l so j o i ne d t o e a c h o t he r by ga p j unc t i ons t ha t c a n be i de n t i f i e d e ve n a t l ow m a gn i f i c a t i on( l a r ge a r r ow s ) , x 10 , 000 .

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FIGURE 5 Freeze-fracture of the eumulus-oocyte junctional contacts in a nonpreovulatory follicle 10 hafter HCG administration. The cumulus processes C) have penetrated the zona pellucida ZP) to imeractboth with one another as well as with the oolemma. In this instance, the fracture process has exposed theouter membrane half E fracture face) of the oolemma O) and the inner membrane half P fracture face)of the cumulus processes that are in junctional contact with the ooeyte. There are two gap junctionalcontacts between cumulus processes and the oolemma that are indicated in this image arrows). 97,200.FIGUaE 6 Freeze-fracture image from a nonpreovulatory follicle showing cumulus processes C) and thesite of a cumulus-oocytegap junction arrow). The inner membrane half P fracture face) of the oolemmaO) has been exposed in this image, x 81,000.

arran gement of pits or depressions Figs 8 and 9).Extensive freeze-fracture images of the oolemmaprovide an illustration of the diversity in form ofcumulus-oocyte interactions Fig. 8). In images ofthe oolemma E fracture-face, bulges in regionsdevoid of microvilli reveal the location of cumuluscell processes that have contacted the oolemma.Almost invariably, gap junctional contacts can bedetected in each of these regions of heterologouscell interaction. Thus, in follicles that are notpreovulatory a large number of small gap junc-tional contacts exist between the oocyte and thecumulus cells.

Gap junctio ns also exist betwee n cumulu s cells.These contacts occur in two locations: a) betw eencumulus cell processes in the region of the zona

pellucida and the oocyte surface Figs. 4 and 6);and b) betwe en cell processes and somatic por-tions of the cumulus cells Figs. t0 a nd 11). Ingeneral, the gap junctions betwee n processes inthe oocyte region are small Fig. 6), while those inthe cumulus area are larger macular structuresFigs. 10 and 11).

In late preovulatory specimens, the number ofcumulus cell processes are significantly reduced.Gap junct ional contacts are still detectable in thecomplex, but they also are reduced in number. Inaddition, the remaining cumulus cells are presentin a loose arrangement with bulbous cytoplasmicextensions Fig. 12). This typical cumulus cellmorphology is retained in the postovulatory spec-imens Fig. 1 D).

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FIGURE 7 Freeze- f racture appeara nce o f the st ructural p leiomo rphism that i s present at the si te ofcumulus-oocyte gap junct ions in a nonpreovulatory fol l ic le . On the inner membrane f racture face, theoolemma is character ized by numerous microvi l l i (M) that are completely absent in the regions of gapjunct ional contact (A-F) . This image contains a representat ive sample of the var ious gap junct ionalpar t icle ar rangements that have been observed. The junct ions may exist as a l inear aggregate of two tothree par t icles (A) , a l inear aggregate of more than a dozen par t icles (B) , a smal l e longated cluster ofpar t icles (C and D) , a y-shaped polygonal aggregate (E) , and a geometr ical ar rangeme nt of polygonallypacked par t icles (F) . 129,600.

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Physiological ObservationsW i t h t h e o b s e r v a t i o n t h a t g a p j u n c t i o n s a r e

p r e s e n t a t a h i g h f r e q u e n c y b e t w e e n c e l ls o f t h ec o r o n a r a d i a t a a n d t h e o o c y t e , i t w a s o f i n t e re s t t od e t e r m i n e w h e t h e r c o m m u n i c a t i o n i n t h e f o r m o fi o n i c c o u p l i n g a n d d y e t r a n s f e r c o u l d b e d e m o n -s t r a t e d b e t w e e n t h e s e c e l l s . F u r t h e r m o r e , s i n c et h e p r o c e s s e s t h a t c o n n e c t t h e t w o c e l l t y p e sa p p e a r t o b e t h e o n l y a v a i l a b le c h a n n e ls f o ri n t e r c e l lu l a r c o m m u n i c a t i o n , a n d s i n c e th e s e p r o c -e s s e s a r e d e g r a d e d n e a r t h e t i m e o f o v u l a t i o n , o n ec o u l d p r e d i c t t h a t c o m m u n i c a t i o n s h o u l d b e d i s -r u p t e d a s o v u l a t i o n a p p r o a c h e s . I n o r d e r t o d e m -o n s t r a t e i n t e r c e l l u l a r c o m m u n i c a t i o n i n t h is sy s -t e m a n d t o d e s c r i b e i t s m o d u l a t i o n , t h e e l e c t r o -p h y s i o l o g i c a l s t u d i es d e s c r i b e d b e l o w w e r e c o n -d u c t e d .

IONIC C OUPLING: M i c r o e l e c t r o d e s w e r er e a d i l y i n s e r t e d i n t o t h e o o c y t e a n d c u m u l u s c e l l s ,a n d re s ti n g m e m b r a n e p o t e n t ia l s o f - 5 0 t o - 6 0t n V w e r e o b t a i n e d o n i n i t i a l p e n e t r a t i o n . W i t ht i m e , t h e r e s t in g m e m b r a n e p o t e n t i a l s d e c l i n e d ,e s p e c i a l ly a f t e r r e p o s i t i o n i n g t h e m i c r o e l e c t r o d e si n t o c u m u l u s c e l ls . U n d e r o u r e x p e r i m e n t a l c o n -d i t i o n s , t h e m e a n r e s t in g p o t e n t i a l o f t h e o o c y t e sw a s - 3 9 - 3 . 1 m V . A s s e e n i n F i g . 1 3 A , t h ei n j e c t i o n o f a c u r r e n t p u l s e i n t o t h e o o c y t e p r o -d u c e d a n e l e c t r o t o n i c p o t e n t i a l r e c o r d e d i n t h ec u m u l u s c e l l , i n d i c a t i n g t h a t t h e t w o c e l l s w e r ei o n i c a ll y c o u p l e d . I n j e c t i o n o f a p u l s e o f c u r r e n ti n t o t h e c u m u l u s c e l l p r o d u c e d a p o t e n t i a l o fs i m i l a r a m p l i t u d e i n t h e o o c y t e , i n d i c a ti n g t h a t t h ei o n i c c o u p l i n g w a s b i d i r e c t i o n a l . S i m i l a r b i d i r e c-t i o n a l c o u p l i n g c o u l d b e d e m o n s t r a t e d b e t w e e nc u m u l u s c e l ls on o p p o s i t e s i d e s o f t h e c o m p l e x( F i gs . 1 3 B a n d D ) .

D Y E T R A N S F E R : C e l l s t h a t c o m m u n i c a t ei o n i c a l l y m a y a l s o tr a n s f e r m o l e c u l e s w i t h m o l e c -u l a r w e i g h t s u p t o 1 , 2 0 0 d a l t o n s ( 2 5 ) . I n o u rs t u d i e s, t h e f l u o r e s c e n t d y e , s o d i u m f l u o r e s c e in( 3 2 3 d a l t o n s ) , w a s u s e d to d e t e r m i n e w h e t h e r t h isf o r m o f c o m m u n i c a t i o n e x i s t s i n t h e c u m u l u s -o o c y t e s y s t e m . W h e n f l u o r e sc e i n w a s io n t o p h o r e t -i c a l l y i n j e c t e d i n t o t h e o o c y t e ( F i g . 14A-D a n dE-H , i t s u b s e q u e n t l y c o u l d b e d e t e c t e d i n t h ec o r o n a r a d i a t a a n d t h e n t h r o u g h o u t t h e c u m u l u so o p h o r u s . U p o n i n s e r ti o n o f t h e f l u o r e s c ei n e l e c-t r o d e i n t o t h e o o c y t e , t h e d y e r a p i d l y f i ll e d ac i r c le , w h i c h c o r r e s p o n d e d i n o u t l i n e t o t h e o o c y t e( F i g . 1 4 B a n d F ) . A f t e r a d e l a y , d y e w a s o b s e r v e di n s o m e o f t h e g r a n u l o s a c e l l s o f t h e c o r o n ar a d i a t a s e p a r a t e d f r o m t h e o o c y t e b y a n u n f i l l e da r e a c o r r e s p o n d i n g t o t h e z o n a p e l l u c i d a ( F i g .1 4 C a n d G ) . I t a p p e a r s f r o m s a m p l e s s u c h as

t h o s e i n F i g . 1 4 C a n d D t h a t s o m e o f t h e c u m u l u sc e l l s r e c e i v e d y e d i r e c t l y f r o m t h e o o c y t e w h i l eo t h e r s m a y r e c e i v e d y e i n d i r e c t l y v ia a d j a c e n tc u m u l u s c e l l s . W i t h d i f f e r e n t o p t i c s o r l o n g e ri n j e c t i o n t i m e s , d y e w a s v i s u a l i z e d in g r a n u l o s ac e l ls p e r i p h e r a l t o t h e c o r o n a r a d i a t a ( F i g . 1 4 Da n d H ) . I f f lu o r e s c e in w a s i n j e c t e d d i r e c t l y in t oc u m u l u s c e l l s , t h e d y e f i l l e d a n u m b e r o f c e ll s( F i g . 1 4 J a n d K ) a n d u l t i m a t e l y ( F i g . 1 4 L ) w a so b s e r v e d i n a l a r g e a r e a a r o u n d t h e o o c y t e . I nt h e s e e x p e r i m e n t s , i t w a s d i f f ic u l t t o d e t e r m i n et h e e x t e n t t o w h i c h f l u o r e s c e i n w a s t r a n s f e r r e d t ot h e o o c y t e b e c a u s e o f t h e l a r g e d i f f e r e n c e i nv o l u m e o f t h e o o c y t e a n d c u m u l u s c e l l s , a n d t h et h r e e - d i m e n s i o n a l a r r a n g e m e n t o f c u m u l u s c e l l sa r o u n d t h e o o c y t e .

TEM POR AL PATTER N OF IONIC C OUPLING IN PR EOVULATOR Y FOLLIC LES: ASs t a t e d a b o v e , o n t h e b a s i s o f m o r p h o l o g i c a l e v i-d e n c e i t a p p e a r e d t h a t c o m m u n i c a t i o n b e t w e e nt h e c u m u l u s a n d t h e o o c y t e m u s t b e i n t e r r u p t e d a ts o m e t i m e n e a r , o r s h o r t l y a f t e r , o v u l a t i o n . I no r d e r t o t e s t t h i s p o s s i b i l i t y , t h e f o l l o w i n g e x p e r i -m e n t w a s c o n d u c t e d . C u m u l u s - o o c y t e p r e p a r a -t i o n s f r o m P M S G - p r i m e d r a t s w e r e e x a m i n e d a tv a r i o u s t i m e s a f t e r t h e a d m i n i s t r a t i o n o f a n o v u -l a t o r y d o s e o f H C G . T h e i m m a t u r e r a t s u s e d int h e s e s t u d i e s o v u l a t e d 1 0 - 1 2 h a f t e r t h e g o n a d o -t r o p i n w a s i n j e c t e d . A s s e e n i n T a b l e I a n d F i g .1 5 , a f t e r th e H C G i n j e c t io n t h e r e w a s a p r o g r e s -s i v e d e c r e a s e i n t h e f r a c t i o n o f p r e o v u l a t o r y c u -m u l u s - o o c y t e p r e p a r a t i o n s t h a t w e r e i o n i c a ll y c o u -p l e d . A t t h e t i m e o f t h e H C G i n j e c t i o n , 1 0 0 o ft h e c u m u l i w e r e c o u p l e d . I n t h e p r e o v u l a t o r yp o p u l a t i o n , t h e f r a c ti o n d e c r e a s e d t o 2 3 n e a rt h e t i m e o f o v u l a t i o n a n d 0 a f t e r o v u l a t i o n . I nc o n t r a s t , c u m u l i f r o m n o n p r e o v u l a t o r y f o l l i c l e sr e m a i n e d c o u p l e d t h r o u g h o u t t h e c o u r s e o f t h ee x p e r i m e n t .

A s s e e n i n T a b l e I , t h e t r a n s f e r r e s i s t a n c e i n t h ep r e o v u l a t o r y s p e c i m e n s d r o p p e d i n t h e h o u r s p r e -c e d i n g o v u l a t i o n . T h i s is c o n s i s t e n t w i t h a d e c r e a s ei n t h e f r e q u e n c y o f i o n i c c o u p l i n g . I t i s a l s oi n t e r e s t i n g t h a t t h e m e m b r a n e p o t e n t i a l o f t h eo o c y t e d e c r e a s e d d u r i n g t h i s s a m e t i m e p e r i o df r o m - 3 9 - 3 . 1 m V ( n = 2 2 ) 1 0 - 1 2 h b e f o r eo v u l a t i o n t o - 2 1 - 2 . 5 m V ( n = 1 2 ) i n p o s t o v u -l a t o r y s p e c i m e n s . A m e a n r e s t i n g m e m b r a n e p o -t e n t i a l o f - 1 4 m V h a s p r e v i o u s l y b e e n r e p o r t e df o r p o s t o v u l a t o r y m o u s e o o c y t e s ( 2 4 ) .Plasminogen Activator Analysis

G r a n u l o s a c e l l s s e c r e t e p l a s m i n o g e n a c t i v a t o rd u r i n g t h e h o u r s p r e c e d i n g o v u l a t i o n ( 6 , 2 7 ) . T h i s

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e n z y m e c o n v e r t s p l a s m i n o g e n , w h i c h i s p r e s e n t i nf o l l i c u l a r f l u i d t o t h e p r o t e a s e p l a s m i n , a t r y p s i n -l i k e e n z y m e w h i c h m i g h t b e c a p a b l e o f d i s r u p t i n gt h e o r g a n i z a t i o n o f t h e c u m u l u s a n d t h u s p l a y ar o l e in th e b r e a k d o w n i n c o m m u n i c a t i o n t h a t w eh a v e r e p o r t e d h e r e .

I n o r d e r t o t e s t t h i s p o s s i b i l i t y , c u m u l u s - o o c y t ep r e p a r a t i o n s f r o m p r e o v u l a t o r y a n d n o n p r e o v u l a -t o r y f o l li c l es w e r e a n a l y z e d f o r s e c r e t e d p l a s m i n -o g e n a c t i v a t o r . A s s e e n i n T a b l e I I , n e i t h e r c l a s sp r o d u c e d d e t e c t a b l e a m o u n t s o f - t h e e n z y m e .

W h i l e t h i s r e s u l t d e c r e a s e s t h e l i k e l i h o o d t h a tp l a s m i n o g e n a c t i v a t o r i s d ir e c t l y r e s p o n s i b le f o rt h e i n te r r u p t i o n o f c o m m u n i c a t i o n i n t h e c u m u l u s ,i t i s i n t e r e s t i n g i n i t s o w n r i g h t s i n c e i t r e v e a l s ac l e a r b i o c h e m i c a l d i f f e r e n c e b e t w e e n g r a n u l o s ac e l ls o f th e c u m u l u s t h a t a r e a s s o c i a t e d w i t h t h eo o c y t e ( c u m u l u s g r a n u l o s a ) a n d t h o s e a s s o c i a t e dw i t h t h e f o l li c le w a ll ( m e m b r a n a g r a n u l o s a ) .

S i n c e t h e a b o v e o b s e r v a t i o n d o e s n o t c o m -p l e t e l y r u l e o u t t h e p o s s i b i l i t y o f a r o l e f o r t h ep l a s m i n o g e n a c t i v a t o r - p la s m i n s y s t e m i n t h e p r o c -

FIGURE 10 Gap junct ional in teract ion between corona radiata cel l s of the cumulus in a nonpreov ulatoryfollicle (thin section), x 81,000.FIGURE 11 Ga p junct ional in teract ion between corona radiata cel l s of the cumulus (freeze-fracture) in anonpreo vulatory fo ll ic le, x 43,740.

F IouaE 8 An ex t ens ive v i ew o f t he ou t e r m em brane f rac t u re face o f t he oo l em m a i n a nonp reovu l a t o ryfol l ic le . At low magnificat ion, the numerous round profi les indicat ing the presence of microviUi are themost obvious feature o f the fracture face. The microvi l l i can also be detected as surface st ructuresextending in to the zona pel lucida Z P , upper right ) . The microvi l l i are conspicuously in terrupted byregions of cumulus-oocyte in teract ions. These regions app ear as convex prot rusions on th is fracture face asa resul t of the cumulus processes indent ing the oocyte surface. At th is magnificat ion, gap junct ionaleleme nts can be detected in the regions of the boutonl ike contacts (arrow) x 15,280.FIGURE 9 Freeze-fractur e mem brane specializations in a nonpreo vulatory fo l l ic le associated wi th thecom pl em en t a ry ou t e r m em brane ha l f (E f rac tu re face ) o f t he oo l em m a. A bank l ike p l aque o f com pl em en-tary gap junct ional p i t s i s present on th is fracture face (arrows) betw een the c lusters of microvi l l i (M ). Aloose arrangement of large part ic les (*) i s frequent ly detected on th is fracture face in c lose proximi ty tothe gap junct ional e lements; these part ic les may be re la ted to the desmosomal contacts that are observedin the th in-sect /on images, x 81,000.

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FxoultE 12 Thin-se ct ion appear ance of the cumul us cel ls in a preovulator y fol l ic le jus t before ovulat ion .Th e ce l ls o f th e co r o n a r ad ia t a a r e s t il l c lo s e to th e zo n a p e l lu c id a ZP), b u t th ey a r e n o lo n g e r a t i g h t lyp ack ed l ay e r o f cu b o id a l ep i th e li a l ce l l s. S o me g ap ju n c t io n s a r e s t il l p r e s en t b e tw ee n th e ce l ls a r r o w s ) ,th e n u mb er o f ce l l p r o ces s es i s g r ea t ly r ed u ced , an d th e ce l ls co n ta in man y b u lb o u s cy to p las mic ex ten s io n son their surfaces . 7 ,460 .

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FIGURE 13 Ionic coupling between no npreovu la tory oocyte and cumulus ce l ls A an d C) and betweencumulus ce l ls B and D). A) The pho tomicrog raph shows an isola ted cumulus with microelec trodesinser ted into the oocyte and a cumulus ce l l. B) The same cumulus prepara t ion as used in A) now withthe microelec trodes inser ted into two cumulus ce l ls . C) A rec tangular pulse of current lower t race) hasbeen injec ted into the oocyte , resul t ing in a br idge def lec t ion middle t race) and an e lec trotonic potentia lrecorded in the cumu lus cel l upper t race) , indica t ive of ionic coupling. A second pulse of current injec tedinto the cumulus ce l l resul ted in an e lec trotonic potentia l middle t race) of s imilar magnitude in theoocyte , indica t ing bidirec t ional ionic coupling. The res t ing potentia ls were - 5 6 mV in the oocyte and -5 6mV in the cumulus ce ll . D) A curren t pulse injec ted into the cumulus ce l l loca ted a t the top of B)produced an e lec trotonic potentia l recorded in the other impaled cumulus ce ll . A second pulse de l iveredto the othe r cumulus ce ll in the lower le f t of B) pro duced a potentia l recorded in the ce l l a t the top ofB) . Re s t ing pote n t ia l s we re -3 8 m V a nd - 40 m V. In both C) a nd D) , the c a librat ion pul se is 5 m V;

vert ica l ca libra t ion l ine is 2.5 x 10 -9 A; horizonta l l ine is 100 ms. The bar in B) represents 50micrometers for both photomicrographs .

e s s o f c u m u l u s - o o c y t e u n c o u p l i n g , c u m u l u s p r e p -a r a t i o n s f r o m P M S G - p r i m e d a n i m a l s w e r e i n cu -b a t e d f o r 2 4 h i n t h e p r e s e n c e o f 1 0 0 / z g / m lp l a s m i n . A f t e r t h i s t r e a t m e n t , t h e p e r i p h e r a l c u -m u l u s c e l l s w e r e l e s s t i g h t l y p a c k e d ; h o w e v e r ,i o n i c c o u p l i n g w a s s t i l l d e t e c t e d b e t w e e n o o c y t ea n d c u m u l u s c e l l s i n e v e r y s a m p l e t h a t w a s a n a -l y z e d . T h u s , t h i s f in d i n g s u g g e s t s t h a t p l a s m i ni t s el f i s n o t r e s p o n s i b l e f o r t h e o b s e r v e d b r e a k -d o w n i n c o m m u n i c a t i o n .

D I S C U S S I O NI n t h i s p a p e r , w e h a v e s h o w n t h a t i n t e r c e l l u l a rc o m m u n i c a t i o n e x i s t s b e t w e e n c e l l s o f t h e c u m u -l u s o o p h o r o u s a i l d t h e o o c y t e . T h e c e l l s a r ei o n i c a l ly c o u p l e d , a n d f l u o r e s c e i n d y e i s r e a d i l yt r a n s f e r r e d b e t w e e n t h e m . T h e c h a n n e l s f o r t h i sc o m m u n i c a t i o n a p p e a r t o b e t h e p r o c e s s e s t h a te m a n a t e f r o m t h e c e l l s o f t h e c o r o n a r a d i a t a a n dt r a v e r s e t h e z o n a p e l l u c i d a . T h e p r o c e s s e s i n t e r a c tw i t h t h e o o l e m m a , a n d g a p j u n c t i o n s a r e p r e s e n t

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F mU R E 1 4 M o v em en t o f i o n to p h o r e t i ca l ly in j ec t ed f lu o r es ce in in th e n o n p r eo v u la to r y cu mu lu s - o o cy teco mp lex . P h o to mic r o g r ap h s o f t h r ee d i f f e r en t cu mu l i a r e s h o w n . I n th e l e f t p an e l A-D), t h e cu mu lu s -o o cy te w as p h o to g r ap h ed w i th p h as e o p t i c s ( A ) , t h en th e o o cy te w as in j ec t ed w i th f lu o r es ce in an ds u b s eq u en t ly p h o to g r ap h e d a f t e r 1 6 r a in ( B ) , 4 8 r a in ( C ) , an d 5 1 r a in ( D ) . O n ly th e o o cy te i s v i s u a l izedin ( B ) , b u t i n ( C ) d y e h as mo v ed in to cu mu lu s ce l l s ad j acen t t o t h e o o cy te w i th o u t o b s cu r in g th e zo n ap e l lu c id a . M an y cu mu lu s ce l l s s h o w f lu o r es cen ce in ( D ) ; a l o n g e r ex p o s u r e w as u s ed in ( D ) th an ( C ) top e r m i t t h e d e t ec t io n o f ce l ls w i th r e la t iv e ly l i t t le f l u o r esce in . M o v em en t i n t h e s ec o n d cu m u lu s o o cy te i ss h o w n i n t h e m i d d l e p a n e l E-H) w i t h p h o t o g r a p h s t a k e n a f t e r 5 r ai n ( F ) , 1 4 r a in ( G ) , a n d 2 5 r a i n ( H ) .I n th e th i r d cu mu lu s o o cy te s h o w n in th e r ig h t p an e l I-L), d y e w as in j ec t ed in to a c u mu lu s ce l l , an d i ts p r ead q u ick ly in to ad jacen t cu m u lu s ce l ls a f t e r 2 r a in ( J ) , 9 m in ( K ) , an d in to m o r e d i s t an t ce l ls a f t e r 1 6min ( L) . C u mu lu s ce l l s l o ca t ed o n to p o f t h e o o cy te a r e f i l l ed w i th f lu o r es ce in an d g iv e th e imp r es s io nth a t d y e h as f i ll ed p a r t o f t h e o o cy te . T h e l i n e in ( L ) r ep r e s en t s 5 0 m ic r o me te r s f o r a ll t h ep h o t o m i c r o g r a p h s .


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I t is o f s o m e i n t e r e s t t o c o n s i d e r t h e r o l e o fc o m m u n i c a t i o n b e t w e e n t h e c u m u l u s a n d t h e o o -c y t e a n d t h e s i g n i f i c a n c e o f i ts i n t e r r u p t i o n a r o u n dt h e t i m e o f o v u l a t i o n . T h e r e p o r t t h a t m u c h m o r e[ a H ] u ri d i ne i s i n c o r p o r a t e d i n t o o o c y t e s w h e n t h e ya r e i n c o n t a c t w i t h t h e c u m u l u s ( 3 0 ) s u g g e s t s t h a tt h e c u m u l u s m a y b e e x t r e m e l y i m p o r t a n t f o ro o c y t e m e t a b o l i s m . F u r t h e r m o r e , s p e ci f ic f a c t o rsi n f l ue n c i n g o o c y t e m a t u r a t i o n m i g h t a l s o b e t r a n s -m i t t e d t o t h e o o c y t e b y i n t e r c e l l u l a r c o m m u n i c a -t i o n . F o r e x a m p l e , i t h a s b e e n r e p o r t e d ( 1 0 , 1 3 )t h a t o o c y t e s i n c r e a s e i n s i z e a s l o n g a s t h e y a r es t il l a t t a c h e d t o t h e c u m u l u s ; g r o w t h i s i n h i b i t e d i ft h e c u m u l u s i s d e t a c h e d o r r e m o v e d .

T h e s i g n if i c a nc e o f a n i n t e r r u p t i o n o f c o m m u -n i c a t i o n n e a r t h e t i m e o f o v u l a t i o n i s n o t c l e a r .O o c y t e s r e m o v e d f r o m a n t r a l fo l li c le s u n d e r g os p o n t a n e o u s r e s u m p t i o n o f m e i os i s . N o r m a l l y t h i so c c u r s o nl y a f t e r t h e p r e o v u l a t o r y L H s u r g e i nv i v o . W h e t h e r t h e r e l i e f o f i n h i b i t i o n is p u r e l y ar e s u lt o f th e o o c y t e b e i n g r e m o v e d f r o m a n e n v i -r o n m e n t c o n t a i n i n g a m e i o s i s i n h i b i t o r y f a c t o r ,s u c h a s t h a t d e s c r i b e d b y T s a f r i r i e t a l. ( 2 9 ) , o r i sa l s o d u e t o a t e r m i n a t i o n o f i n t e rc e l l u l a r c o m m u -n i c a t i o n w i t h t h e c u m u l u s m u s t b e e x a m i n e df u r t h e r .W e wi sh t o t h a n k Jo a n Pe se k a n d E le a n a Sp h ic a s f o re x c e l l e n t te c h n i c a l a s s i s t a n c e , Asn e th K lo e sm a n f o r h e lpin p r e p a r in g t h e f i g u r e s , M a d e l e in e Na y lo r f o r se c r e t a r i a la s s i s t a n c e , a n d D r . N a v a D e k e l f o r h e lp f u l d i scu ss io n s .

T h i s wo r k wa s su p p o r t e d b y g r a n t s f r o m T h e R o c k e -f e l l e r Fo u n d a t io n ( R F 7 0 0 9 5 ) , T h e Na t io n a l I n s t i t u t e o fC h i l d H e a l t h a n d H u m a n D e v e l o p m e n t ( H D - 0 9 4 0 1 ) ,T h e N a t i o n a l H e a r t a n d L u n g I n s t it u t e (H L 1 6 5 0 7 ) , a n dth e I r m a T . H i r sc h l T r u s t . N . B . G i lu l a a n d W . H . B e e r sa r e r e c ip i e n t s o f Un i t e d S t a t e s Pu b l i c He a l t h Se r v i c eR e s e a r c h C a r e e r D e v e l o p m e n t A w a r d s ( H L - 0 0 1 1 0 a n dH D - 0 0 1 8 9 ) .R e c e i v e d f o r p u b l i c a t io n 1 7 N o v e m b e r 1 9 7 7 , a n d i nr e v i se d fo r m 1 0 F e b r u a r y 1 9 7 8 .

R E F E R E N C E S1. AMSTERDAMA. , Y . Ko c H, M . E . L i e b e r m a n , a n d

H. R . LINDNER. 1975 . Dist r ibu t ion of b ind ing si tesf o r h u m a n c h o r io n i c g o n a d o t r o p in i n t h e p r e o v u la -to ry fo l lic le o f the ra t . J . C e l l B io l . 6 7 : 8 9 4 - 9 0 0 .

2. AMSTERDAM A. R. JOSEPHS M. E. LIEBERMAN,a n d H . R . L INDNER . 1 9 7 6 . Or g a n i z a t i o n o f i n t r a -m e m b r a n e p a r t i c l e s i n f r e e z e - c l e a v e d g a p j u n c t i o n sof ra t g raaf ian fo l l ic les: op t ica l -d i f f rac t ion analysis .J . C e l l Sc i . 2 1 : 9 3 - 1 0 5 .

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