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Ultrastructure of the rat ovarian germinal epithelium and its permeability to electron microscopically.. Donaldson, Ranald Ross 1976

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THE  ULTRASTRUCTURE OF THE RAT OVARIAN GERMINAL EPITHELIUM  AND ITS PERMEABILITY TO ELECTRON MICROSCOPICALLY DEMONSTRABLE TRACER MOLECULES by RANALD ROSS DONALDSON B.Sc,  University of V i c t o r i a ,  1973  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE  REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  i n the Department o f ANATOMY  We accept t h i s t h e s i s as conforming to the r e q u i r e d  THE  UNIVERSITY OF BRITISH COLUMBIA April,  ©  standard  1976  Ranald Ross Donaldson, 1976  In p r e s e n t i n g t h i s  thesis  an advanced degree at the I  Library shall  fulfilment of  the requirements f o r  the U n i v e r s i t y of B r i t i s h Columbia,  make i t  freely available  f u r t h e r agree t h a t p e r m i s s i o n  for  for  I agree  r e f e r e n c e and  f o r e x t e n s i v e copying o f  this  that  study. thesis  s c h o l a r l y purposes may be granted by the Head o f my Department or  by h i s of  in p a r t i a l  this  representatives. thesis  It  is understood that copying or p u b l i c a t i o n  f o r f i n a n c i a l gain s h a l l  written permission.  Department of The  ANATOMY  University of B r i t i s h  2075 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1W5  Date  A p r i l 6.  1976  Columbia  not be allowed without my  i i  ABSTRACT The d e t a i l e d f i n e s t r u c t u r e o f normal r a t o v a r i a n germinal e p i t h e l i u m was s t u d i e d by means o f t r a n s m i s s i o n and scanning e l e c t r o n microscopy.  The germinal e p i t h e l i u m possesses f e a t u r e s  s u g g e s t i v e o f an a b s o r p t i v e and/or s e c r e t o r y c a p a c i t y , a marked p r o t e i n s y n t h e t i c a b i l i t y , and p o s s i b l e s t e r o i d  metabolism.  These and o t h e r c e l l u l a r f e a t u r e s were i n t u r n i n d i c a t i v e o f a p o s s i b l e g e r m i n a l e p i t h e l i a l involvement i n t r a n s c e l l u l a r movement o f p a r t i c u l a t e  substances.  In order t o i n v e s t i g a t e the p e r m e a b i l i t y o f the g e r m i n a l e p i t h e l i u m t o molecules from the p e r i t o n e a l c a v i t y , two e l e c t r o n m i c r o s c o p i c t r a c e r molecules, h o r s e r a d i s h peroxidase (HRP) and f e r r i t i n , were i n j e c t e d i n t r a p e r i t o n e a l l y .  The r e s u l t s  indicate  t h a t there i s a d i f f e r e n t i a l movement o f these two molecules a c r o s s the g e r m i n a l e p i t h e l i u m , presumably  r e l a t e d t o the  d i f f e r e n c e ; i n t h e i r m o l e c u l a r dimensions.  The  predominant  r o u t e o f movement o f HRP i s e x t r a c e l l u l a r , a p p a r e n t l y by d i f f u s i o n through the i n t e r c e l l u l a r c l e f t s .  Ferritin movement, on (l  the o t h e r hand, i s i n t r a c e l l u l a r , v i a a v e s i c u l a r t r a n s p o r t mechanism a s s o c i a t e d with p i n o c y t o t i c a c t i v i t y a t the a p i c a l s u r f a c e o f the germinal e p i t h e l i a l  cells.  I t i s concluded t h a t the g e r m i n a l e p i t h e l i u m i s a metab o l i c a l l y a c t i v e t i s s u e which p l a y s both a p a s s i v e and an a c t i v e r o l e i n the movement o f molecules from the p e r i t o n e a l cavity.  iii  TABLE OF CONTENTS Page ABSTRACT  i i  TABLE OF CONTENTS  i i i  LIST OF FIGURES  v  ACKNOWLEDGEMENT  vi  INTRODUCTION  1  A. P r e v i o u s S t u d i e s o f the Germinal E p i t h e l i u m  ....  1  B. E l e c t r o n M i c r o s c o p i c Tracers  5  C. Scope o f the Present Study  .  9  MATERIALS AND METHODS  11  RESULTS  18  A. Normal Germinal E p i t h e l i u m  18  1. General E p i t h e l i a l Morphology 2. C e l l Membrane Features a. L a t e r a l c e l l membranes* clefts  and j u n c t i o n s  18 j  19  intercellular ••••  1  9  b. A p i c a l c e l l membrane  21  c. B a s a l c e l l membrane  22  3. Nuclear  Structure  4. O r g a n e l l a r S t r u c t u r e B. T r a c e r Experiments  22 23 25  1. HRP D i s t r i b u t i o n  25  2. F e r r i t i n D i s t r i b u t i o n  30  C. F i g u r e s  34  iv  Table o f Contents  (Cont.) Page  DISCUSSION  5  5  A. Germinal E p i t h e l i a l C e l l Shape  55  B. I n t e r c e l l u l a r J u n c t i o n s  57  C . Microvilli  ^  60  D. P r o t e i n S y n t h e s i s  62  E. L i p i d s  64  •  F. HRP Movement and L o c a l i z a t i o n  66  G. F e r r i t i n Movement and L o c a l i z a t i o n  68  CONCLUSION LITERATURE CITED  72 ,  76  V  LIST OF FIGURES  Figures  Pages  1-11  Normal germinal e p i t h e l i u m  35-42  12-18  Germinal e p i t h e l i u m exposed t o HRP  4 3-48  19-23  Germinal e p i t h e l i u m exposed t o  49-54  ferritin  vi  ACKNOWLEDGEMENT To D r . W. A. Webber, my s u p e r v i s o r d u r i n g t h e c o u r s e o f this  study, I would l i k e  guidance,  t o e x p r e s s my s i n c e r e g r a t i t u d e .  encouragement, and s u p p o r t t h r o u g h o u t  His  a l l stages of  the s t u d y were g r e a t l y a p p r e c i a t e d . I am a l s o i n d e b t e d t o D r s .  W. K. O v a l l e , B. J . P o l a n d , a n d  M. E. Todd f o r t h e i r p a i n s t a k i n g a n d t h o r o u g h  c r i t i c i s m s and  valuable suggestions during the.preparation of t h i s F i n a l l y , my t h a n k s go t o M r s .  thesis.  Patricia Hollingdale,  whose e x p e r t t e c h n i c a l a s s i s t a n c e i n a l l p h a s e s o f t h i s it  c o u l d n o t have been  without project,  completed.  T h i s s t u d y was s u p p o r t e d b y a S t u d e n t s h i p f r o m t h e M e d i c a l Research  C o u n c i l o f Canada.  1  INTRODUCTION A.  Previous  S t u d i e s o f the Germinal E p i t h e l i u m  The f u n c t i o n a l s i g n i f i c a n c e o f the germinal the mammalian ovary was f i r s t  e p i t h e l i u m of  i n v e s t i g a t e d i n terms o f i t s  p o s s i b l e oogenic p o t e n t i a l (Waldeyer, 1870, quoted by F r a n c h i et  a l . , 1962).  As c l e a r l y e v i d e n t by the name accorded i t by  e a r l y workers, the germinal source  e p i t h e l i u m was c o n s i d e r e d  o f o v a r i a n germ c e l l s , ( F r a n c h i e t a l . , 1962).  t o be a Subse-  quent s t u d i e s have demonstrated t h a t p r i m o r d i a l germ c e l l s which g i v e r i s e t o d e f i n i t i v e oocytes  a r i s e e i t h e r from stem  c e l l s which give r i s e t o endodermal c e l l s , or from the endoderm o f the embryonic secondary y o l k s a c , near the s i t e of the a l l a n t o i c evagination  ( W i t s c h i , 19#8).  These germ c e l l s  then  a c t i v e l y migrate i n an amoeboid f a s h i o n , perhaps a s s i s t e d by h i s t i o l y t i c a c t i o n , t o the r e g i o n o f the gonadal blastema by way o f the d o r s a l mesentery o f the developing 19^8} tially  P i n k e r t o n e t a l . , 1961).  The gonadal blastema i s i n i -  a t h i c k e n e d r e g i o n o f the coelomic  u n d e r l y i n g mesenchyme on the ventromedial urogenital ridges. epithelium.  gut ( W i t s c h i ,  mesothelium  and  aspect o f the  This mesothelium l a t e r becomes the germinal  I t i s now w e l l e s t a b l i s h e d t h a t a l l d e f i n i t i v e  germ c e l l s are progeny of the p r i m o r d i a l germ c e l l s which first  populate  the ovary.  The germinal  e p i t h e l i u m does not  c o n t r i b u t e t o the germ c e l l  p o p u l a t i o n a t any stage o f o v a r i a n  development ( W i t s c h i , 19631  Franchi,  1970).  C u r r e n t l y , a number o f i n v e s t i g a t o r s b e l i e v e t h a t the  2  germinal e p i t h e l i u m  i s a c t i v e mainly d u r i n g p r e n a t a l ,  and  perhaps e a r l y p o s t n a t a l development.  At these times i t i s  b e l i e v e d t o be a source o f p r e c u r s o r s  of f o l l i c u l a r  granulosa) c e l l s of the d e v e l o p i n g 1970).  ovarian cortex  This i s d i s p u t e d , however, by others who  (later,  (Franchi, suggest t h a t  the f o l l i c u l a r c e l l s are d e r i v e d from mesenchymal and mesothelial Apart  (germinal e p i t h e l i a l ) c e l l s  not  ( F r a n c h i , 197°)•  from the developmental f u n c t i o n o f the germinal  epithelium,  i t s f u n c t i o n a l a c t i v i t y i n the p o s t n a t a l animal ( e x c l u s i v e of e a r l i e r s t u d i e s of p o t e n t i a l germinal a c t i v i t y ) has not been seriously  considered.  Most r e c e n t s t u d i e s o f the g e r m i n a l e p i t h e l i u m have been concerned with  i t s d e s c r i p t i v e u l t r a s t r u e t u r e , both  during  i n t r a - u t e r i n e and p o s t n a t a l development ( W i s c h n i t z e r , Gondos, 1969i Weakley, 1969» Papadaki and B e i l b y , Jeppesen, 1975? Merchant, 19751 and  P e l l i n i e m i , 1975).  19651  1971; Papadaki  B e i l b y (1971) and Weakley (1969) have suggested t h a t  germinal e p i t h e l i a l c e l l s , f e a t u r e s , may  on the b a s i s o f t h e i r u l t r a s t r u e t u r a l  t r a n s f e r m a t e r i a l through the cytoplasm.  The  d i r e c t i o n of movement, however, c o u l d not be a s c e r t a i n e d from t h i s morphological  data alone.  c y t o t i c v e s i c l e s , vacuoles,  These workers observed  m i c r o v i l l i and an abundance o f  ribosomes, rough endoplasmic r e t i c u l u m , and m i t o c h o n d r i a the germinal e p i t h e l i a l c e l l s .  Since such f e a t u r e s are  prominent i n c e l l s able t o c a r r y on t r a n s p o r t and processes,  they c o n s i d e r e d  pino-  within often  synthetic  t h e i r assumptions warranted.  Of those s t u d i e s of the germinal e p i t h e l i u m conducted to  3  date, o n l y Ghiquoine's work (1961) i n v o l v e d an experimental component i n an u l t r a s t r u e t u r a l study ( l i g h t m i c r o s c o p i c s t u d i e s w i l l be mentioned l a t e r ) .  To determine whether o r  not the germinal e p i t h e l i u m had an oogenic f u n c t i o n , mice were i n j e c t e d i n t r a p e r i t o n e a l l y with c o l l o i d a l g o l d , an e l e c t r o n m i c r o s c o p i c a l l y demonstrable  t r a c e r substance.  I f the germinal  e p i t h e l i a l c e l l s were l a b e l l e d with g o l d and gave r i s e t o germ cells  (which have d i s t i n c t l y r e c o g n i z a b l e h i s t o l o g i c a l ,  histo-  c h e m i c a l , and u l t r a s t r u c t u r a l f e a t u r e s ) , then Chiquoine would have expected t o f i n d p a r t i c u l a t e g o l d t r a c e r w i t h i n the c e l l s so d e r i v e d .  Because no oocytes were l a b e l l e d , Chiquoine  con-  c l u d e d t h a t " v i t a l s t a i n i n g o f the germinal e p i t h e l i u m p r o v i d e s no evidence f o r an oogenic f u n c t i o n on the p a r t o f the germinal e p i t h e l i u m i n the p o s t n a t a l animal."  This r e s o l v e d the con-  f l i c t i n g r e s u l t s of e a r l i e r l i g h t microscopic tracer studies ( L a t t a and Pederson,  1944j Jones, 19^9), which were l i m i t e d i n  p a r t by v i r t u e o f i n h e r e n t t e c h n i c a l r e s t r i c t i o n s .  Chiquoine's  d e s c r i p t i o n o f t r a c e r l o c a l i z a t i o n was b r i e f , s t a t i n g t h a t aggregates o f g o l d p a r t i c l e s were randomly s c a t t e r e d w i t h i n the germinal e p i t h e l i a l c e l l s .  No mention was made o f how the g o l d  t r a c e r might have e n t e r e d the c e l l s , nor whether i t s course was f o l l o w e d over a p e r i o d o f time u s i n g a s e r i e s o f animals. Zuckerman (195D has a l s o made mention  o f the uptake of  p a r t i c u l a t e matter by the germinal e p i t h e l i u m .  He s a i d t h a t  i t " i s h i g h l y p h a g o c y t i c , and i n the normal animal p i c k s up d e b r i s from r e d blood c e l l s or any p a r t i c u l a t e matter o f s u i t a b l y s m a l l s i z e t h a t i s i n t r o d u c e d i n t o the p e r i t o n e a l  4  cavity." evidence  Unfortunately,  t h i s paper presented  no s u b s t a n t i a t i n g  f o r t h a t statement, nor d i d i t mention the experimental  animal t o which t h a t statement r e f e r r e d . Chiquoine's  study,  although  presumably n o t intended t o  i l l u s t r a t e t r a c e r movement across the germinal did  show t h a t germinal  gold.  epithelium,  e p i t h e l i a l c e l l s c a n take up c o l l o i d a l  T h i s uptake c o u l d r e p r e s e n t the f i r s t step o f a c e l l -  mediated t r a n s p o r t .  However, s i n c e then there have been no  r e p o r t s i n the l i t e r a t u r e concerned with t r a c e r movement across the germinal  epithelium.  Such movement c o u l d c o n c e i v a b l y be  e i t h e r by p a s s i v e d i f f u s i o n or by a c t i v e t r a n s p o r t . occur across the germinal  epithelium i n a  I t could  u n i d i r e c t i o n a l or  b i d i r e c t i o n a l fashion, e i t h e r i n t r a c e l l u l a r l y or e x t r a c e l l u l a r l y . Any  such t r a n s p o r t p r o p e r t i e s c o u l d be o f p o t e n t i a l p h y s i o l o g i c a l  and  developmental s i g n i f i c a n c e t o the ovary.  e s p e c i a l l y t o the oocytes  This a p p l i e s  i n t h a t c e r t a i n substances,  perhaps of a d e l e t e r i o u s nature  some  i n terms of oocyte development,  might be able t o pass o r be t r a n s p o r t e d from the p e r i t o n e a l c a v i t y i n t o the substance o f the ovary. Transport  c o u l d a l s o be o p e r a t i v e over s l i g h t l y  shorter  d i s t a n c e s i n the case o f substances a c t u a l l y manufactured w i t h i n the germinal  e p i t h e l i a l c e l l s and t r a n s f e r r e d t o sub-  j a c e n t c e l l s o f the t u n i c a a l b u g i n e a o v a r i i and c o r t e x . Weakley (1969) suggests the p o s s i b i l i t y o f r e c i p r o c a l i n d u c t i o n by mutual t r a n s f e r o f substances, p r o t e i n s , between the germinal follicle cells  such as amino a c i d s and  e p i t h e l i a l c e l l s and u n d e r l y i n g  i n e a r l y development.  5  Accordingly, microscopic  t r a c e r s were chosen i n an attempt to examine  characterize  the germinal e p i t h e l i u m  and/or p a s s i v e B.  i n the present study, exogenous e l e c t r o n  transport  E l e c t r o n Microscopic The  use  and  i n terms o f i t s a c t i v e  properties. Tracers  of electron microscopically  demonstrable t r a c e r s  t o e l u c i d a t e pathways o f normal molecular uptake, t r a n s f e r , r e l e a s e from c e l l s has years.  Currently  become of i n c r e a s i n g value i n r e c e n t  used t r a c e r s r e p r e s e n t  o f molecular s i z e and  and  weight, and  a considerable  range  as such c o n s t i t u t e a graded  s e r i e s of m o l e c u l a r probes with which c e l l s and t i s s u e s may investigated.  In both q u a l i t a t i v e and q u a n t i t a t i v e  be  determination  of molecular movement c h a r a c t e r i s t i c s , s e l e c t i o n o f a t r a c e r depends a l s o on a number of o t h e r important f a c t o r s . the dosage r e q u i r e d s h o u l d not be i n i m i c a l to the normal p h y s i o l o g i c a l f u n c t i o n i n g o f the animal.  Firstly,  continued A l s o , molecules  o f the t r a c e r should be of a uniform s i z e , so as to  allow  accurate assessment and q u a n t i f i c a t i o n o f r e s u l t s .  In a d d i t i o n ,  the t r a c e r s h o u l d not be m e t a b o l i z e d by  the t i s s u e s nor  should  c l a s s i f i e d into  two  i t be r a p i d l y c l e a r e d from the body. E l e c t r o n microscopic main groups.  The  t r a c e r s may  be  f i r s t c o n s i s t s of molecules which are  able by v i r t u e o f t h e i r n a t u r a l e l e c t r o n o p a c i t y .  detect-  Once h a v i n g  exposed t i s s u e s t o t h i s type of t r a c e r , r o u t i n e p r o c e s s i n g  and  examination w i l l r e v e a l the presence (or absence) of these molecules.  Tracers  o f t h i s type may  or n o n - b i o l o g i c a l o r i g i n .  be  of e i t h e r b i o l o g i c a l  They are e x e m p l i f i e d  by  lanthanum  (Revel and Karnovsky, (Leak, 1971). f e r r i t i n  1967), carbon (Leak, 1971). l a t e x spheres (see p. 8 ), dextrans (Simionescu and  Palade, 1971). glycogen (Simionescu  and Palade, 1971). and  c o l l o i d a l suspensions o f g o l d (Ghiquoine, 1961) and mercuric s u l p h i d e (Odor, 1956) . The second group o f t r a c e r s i s enzymatic  i n nature.  s i t e of t h e i r l o c a l i z a t i o n w i t h i n a t i s s u e or c e l l o n l y subsequent  t o the exposure  The  is visible  o f the t i s s u e t o a s u b s t r a t e  a p p r o p r i a t e t o the p a r t i c u l a r enzyme used as a t r a c e r .  What  i s then seen i s the e l e c t r o n m i c r o s c o p i c a l l y v i s i b l e end product of  the r e a c t i o n , which should be l o c a l i z e d as a p r e c i p i t a t e  w i t h i n the immediate v i c i n i t y o f the enzyme. of  T r a c e r molecules  the second type must be by d e f i n i t i o n o f b i o l o g i c a l  origin,  and are mainly p e r o x i d a t i c enzymes 1 h o r s e r a d i s h peroxidase (see  p. 8 ), myeloperoxidase  (Graham and Karnovsky,  (HRP)  1966a),  l a c t o p e r o x i d a s e (Graham and Kellermeyer, 1968), microperoxidase (Feder, 1970), c a t a l a s e (Goodenough and Revel, 1971). (Anderson, W.A., I969).  myoglobin  1972b), and cytochrome c (Karnovsky and R i c e ,  E x p e r i m e n t a l l y the s i t e s of l o c a l i z a t i o n of these enzymes  w i t h i n a t i s s u e or c e l l are t y p i c a l l y v i s u a l i z e d v i a a r e a c t i o n i n v o l v i n g hydrogen peroxide and 3,3'-diaminobenzidine (Graham and Karnovsky,  1966b; H i r a i , 1975).  the r e a c t i o n sequence o f HRP with H 0 2  ( m o d i f i e d a f t e r White e t a l . ,  1973).  (DAB)  As an example  and DAB i s shown below  2  To show t h a t DAB i s  i n i t i a l l y a hydrogen donor i n the f o l l o w i n g r e a c t i o n , i t i s r e p r e s e n t e d as DABHg. HRP-H 0 + H 0 2  2  2  >-  HRP-H 0 2  (complex  2  + HgO I)  7 HRP-H 0 2  2  + DABH  >-  2  complex I I + DABH -  complex I I + DABH • >• HRP-HgO +  The r e a c t i o n sequence i n v o l v e s  DAB  the f o r m a t i o n o f c o n s e c u t i v e  complexes which have not y e t been p r e c i s e l y d e f i n e d .  The  a l l r e s u l t o f the r e a c t i o n i s the r e g e n e r a t i o n o f HRP, w i l l continue to c a t a l y z e o f o x i d i z e d DAB,  over-  which  the same r e a c t i o n , and the f o r m a t i o n  which forms an i n s o l u b l e p r e c i p i t a t e a t the  s i t e s o f r e a c t i o n (Seligman e t a l . , 1968).  The p r e c i p i t a t e is=  a brown pigment a t the l i g h t m i c r o s c o p i c l e v e l and an e l e c t r o n opaque substance a t the e l e c t r o n m i c r o s c o p i c The aforementioned  level.  c r i t e r i a f o r choosing t r a c e r s , as w e l l  as the s u c c e s s f u l experience of o t h e r workers with c e r t a i n o f these t r a c e r s , were taken i n t o c o n s i d e r a t i o n  when choosing t r a c e r  molecules with which to i n v e s t i g a t e the germinal e p i t h e l i u m . I t was  decided to use two t r a c e r molecules, f e r r i t i n and  By v i r t u e o f t h e i r r e s p e c t i v e molecular dimensions  and  HRP.  weight  they would be expected to have d i f f e r e n t t r a n s f e r c h a r a c t e r i s t i c s , and thus bound a s i z e a b l e range of p o s s i b l e t r a c e r s which c o u l d be  used. F e r r i t i n , the l a r g e r o f the two t r a c e r s , i s a heme p r o t e i n  containing  20-24% i r o n which was  f i r s t i s o l a t e d from  s p l e e n (Ainsworth and Karnovsky, 1971).  The molecule  horse consists  of a s p h e r i c a l s h e l l of p r o t e i n s u r r o u n d i n g a core of f e r r i c o  hydroxide m i c e l l e s .  The t o t a l molecular diameter  i s 110  A,  o  whereas the core diameter i s approximately 55 A (Ainsworth Karnovsky, 1971). molecule.  and  The core i s the e l e c t r o n dense p a r t o f the  In o r d e r to a v o i d m i s i n t e r p r e t a t i o n  of r e s u l t s i t  8  must t h e r e f o r e be kept i n mind t h a t the e n t i r e molecule i s not seen i n e l e c t r o n micrographs. f e r r i t i n i s about  462,000 (Ainsworth and Karnovsky,  The second t r a c e r , HRP, tracer studies.  The molecular weight  1971).  has a l s o been e x t e n s i v e l y used i n  The reason f o r the e f f i c a c y of t h i s  i s a p t l y d e s c r i b e d by Graham and Karnovsky method i s s e n s i t i v e because  of  enzymatic  (1966b)t  tracer "the  a c t i v i t y has an a m p l i -  f y i n g e f f e c t j thus a few molecules o f p r o t e i n a t a s i t e  can  generate a much l a r g e r amount o f r e a c t i o n product upon i n c u b a o  tion."  HRP  has a molecular diameter o f about 40 A and a  molecular weight of about 40,000 (Klapper and Hackett, 1965). Commercially  a v a i l a b l e HRP  c o n t a i n s a number of components  which are separable by s t a r c h e l e c t r o p h o r e s i s . composition, a b s o r p t i o n spectrum, of  The amino a c i d  s i z e , and enzymatic  activity  these components are a l l q u i t e s i m i l a r (Klapper and Hackett,  1965).  C a t a l y t i c a c t i v i t y of the f i v e p u r i f i e d peroxidase  f r a c t i o n s was  i d e n t i c a l i n the two assays c a r r i e d out by  Klapper and Hackett (1965).  They concluded t h a t the t o t a l  enzymatic a c t i v i t y o f u n f r a c t i o n a t e d HRP  would not be  affected  by d i f f e r e n t r e l a t i v e amounts o f the f r a c t i o n s . HRP  and f e r r i t i n have been used as t r a c e r s i n a number of  d i f f e r e n t experimental s i t u a t i o n s .  One  or other or both of  them have been used t o study the p e r m e a b i l i t y o f c a p i l l a r i e s i n c a r d i a c and s k e l e t a l muscle (Karnovsky and Cotran, 19665 Karnovsky,  1967? Bruns and Palade, 1968), lung  Keeley and Karnovsky,  (Schneeberger-  19685 Clementi, 1970), i n t e s t i n e (Clementi  and Palade, 1969). cerebrum (Reese and Karnovsky,  1967),  9  thymus ( G e r v i n and Holtzman, 1972), ovary (Anderson, Payer, 1975). and r e n a l glomerulus Farquhar e t a l . ,  W.,  (Farquhar and Palade,  1961; Webber and Blackbourn, 1970).  1972a; 1961;  They have  a l s o been used t o study p e r m e a b i l i t y and a b s o r p t i o n i n proximal r e n a l tubule (Graham and Karnovsky, mesothelium (Karnovsky  1966b; Maunsbach, 1966),  and Cotran, 1966; C o t r a n and  Karnovsky,  1968; Kluge, 1969). p e r i c a r d i u m (Kluge and Hovig, 1968; 1969). o v a r i a n f o l l i c l e oocyte  (Anderson,  W.,  1972a; Payer,  Kluge,  1975).  (Anderson, S., 1967. 1972), p a r i e t a l l a y e r o f Bowman's  c a p s u l e (Webber and Blackbourn, 1971). p o s t - o v u l a t o r y zona p e l l u c i d a (Hastings e t a l . ,  1972), u r i n a r y b l a d d e r e p i t h e l i u m  (Wade and D i s c a l a , 1971), and c u l t u r e d and normal tumor c e l l s (Ryser e t a l . ,  1962).  F e r r i t i n can a l s o be l i n k e d , u s i n g b i f u n c t i o n a l c o n j u g a t i n g agents, to antibody, i n o r d e r t o l o c a l i z e s i t e s o f a n t i g e n antibody r e a c t i o n s .  P r o t e i n compounds l a b e l l e d with  have i n c l u d e d a n t i f i b r i n o g e n (Wylie, 1964), enzymes et a l . , G.  1966), and a n t i v i r a l g l o b u l i n (Morgan e t a l . ,  Scope o f the Present  ferritin (Benjaminson 1961).  Study  A d e s c r i p t i o n o f the normal u l t r a s t r u e t u r e o f p r e - and p o s t p u b e r t a l r a t germinal e p i t h e l i u m w i l l be presented, as the e p i t h e l i u m o f t h i s s p e c i e s has n o t y e t been adequately d e s c r i b e d i n the l i t e r a t u r e  i n terms o f i t s f i n e  structure.  The p r e s e n t study i s a l s o concerned with the movement o f two t r a c e r s , HRP and f e r r i t i n ,  from the p e r i t o n e a l c a v i t y i n t o and  a c r o s s the germinal e p i t h e l i u m .  By o b s e r v i n g the l o c a l i z a t i o n  o f these t r a c e r s w i t h the pated that the  the  e l e c t r o n microscope,  a c t i v e and/or passive  germinal epithelium  m i g h t be  transport  elucidated.  i t was  antici-  capacities  of  11  MATERIALS AND METHODS Female a l b i n o r a t s of the Wistar s t r a i n were used i n t h i s study.  They ranged i n age  from 29 days to 1? months, thus  r e p r e s e n t i n g both p r e - and p o s t p u b e r t a l animals. Rat Chow) and water were p r o v i d e d ad l i b i t u m .  Food ( P u r i n a  A d u l t animals  were s e l e c t e d without r e g a r d to the exact stage o f the  ovarian  cycle. HRP  (type I I , Sigma Chemical Company, S t . L o u i s , M i s s o u r i )  and f e r r i t i n  (horse s p l e e n , 2X c r y s t a l l i n e , cadmium f r e e ,  N u t r i t i o n a l Biochemical C o r p o r a t i o n , C l e v e l a n d , Ohio, o r Pharmaceuticals,  ICN  Inc., L i f e Sciences Group, C l e v e l a n d , Ohio)  were chosen as the e l e c t r o n m i c r o s c o p i c t r a c e r s . i n dosages v a r y i n g from 8-100  HRP  was  used  mg/100 g body weight and i n  c o n c e n t r a t i o n s r a n g i n g from 10-40  mg/ml i s o t o n i c  saline.  F e r r i t i n dosages of 20-200 mg/100 g body weight, a t t i o n s o f 10-100 mg/ml i s o t o n i c s a l i n e , were used. r e c e i v e d an i n t r a p e r i t o n e a l i n j e c t i o n o f one  concentraAnimals  or other o f the  t r a c e r s i n the r i g h t lower quadrant of the abdomen.  After  s p e c i f i c p e r i o d s of time these animals were a n e s t h e t i z e d  and  sacrificed.  and  In a d d i t i o n , some animals were a n e s t h e t i z e d  t h e i r o v a r i e s d i r e c t l y immersed i n f e r r i t i n a t a c o n c e n t r a t i o n o f 100  mg/ml i s o t o n i c s a l i n e .  In e i t h e r case the a n e s t h e t i c  c o n s i s t e d o f a s e q u e n t i a l combination  of i n t r a p e r i t o n e a l l y  i n j e c t e d sodium p e n t o b a r b i t a l ( c o n c e n t r a t i o n ! 0.2  ml/100 g) f o l l o w e d by subcutaneously  phenobarbital  (concentration!  Animals i n j e c t e d with HRP  Z,0%i  3-3$s dosage i  i n j e c t e d sodium  dosagei  0.2  ml/100 g ) .  were s a c r i f i c e d a t 45 min,  65  min,  12  2 hr, 4 hr, and 5 h r .  P o s t - i n j e c t i o n s a c r i f i c e times f o r  animals r e c e i v i n g f e r r i t i n i n j e c t i o n s were 30 min, 45 min, 1 hr, 2 hr, 4 h r , 4§ h r , and 24 h r . times f o r a n e s t h e t i z e d  Post-immersion  sacrifice  animals with t h e i r o v a r i e s immersed i n  f e r r i t i n s o l u t i o n were 15 min, 30 min, 1 hr, l i hr, 2 h r , and  3* h r . To expose the o v a r i e s , a m i d l i n e  i n c i s i o n was made through  the v e n t r a l abdominal w a l l i n t o the p e r i t o n e a l c a v i t y from a p o s i t i o n j u s t r o s t r a l t o the v a g i n a l opening as f a r as the subcostal  line.  F i x a t i v e was immediately  introduced  i n t o the  p e r i t o n e a l c a v i t y a t t h i s p o i n t i n those animals which had received p r i o r injections of tracer.  L a t e r a l i n c i s i o n s were  then made f o r approximately 1 cm on e i t h e r side o f the c a u d a l end o f the f i r s t  incision.  The abdominal w a l l f l a p s so c r e a t e d  were t h e n r e t r a c t e d and most o f the s m a l l i n t e s t i n e d i s p l a c e d to the o u t s i d e the o v a r i e s . the u t e r u s ,  o f the abdominal c a v i t y .  This manoeuvre  exposed  Each was s i t u a t e d near the end o f each horn o f s e p a r a t e d from i t by the h i g h l y c o i l e d  oviduct.  A tendinous band, t r a v e l l i n g w i t h i n the broad ligament from the d o r s a l a s p e c t o f each ovary to a p o i n t on the d o r s a l body w a l l a d j a c e n t t o the i p s i l a t e r a l diaphragmatic c r u s , each ovary i n p o s i t i o n . oviduct,  maintained  This band was c u t and the u t e r i n e  horn,  and ovary r a i s e d o f f the d o r s a l body w a l l so t h a t the  ovary, c o n t a i n e d  w i t h i n i t s bursa, c o u l d be removed.  The r e l a t i o n s h i p o f the o v i d u c a l mesenteries t o the mammalian ovary i s h i g h l y v a r i a b l e (Beck, 1972). anatomic  independence  There may be complete  o f these s t r u c t u r e s , with much o f the  13 s u r f a c e of the ovary f r e e l y exposed, such as i s seen i n the human and deer (Beck, 1972).  On the other hand the ovary  may  be completely surrounded by a c l o s e d b u r s a d e r i v e d from  the  o v i d u c a l mesenteries, such as t h a t seen i n the golden  hamster (Clewe, 1966).  The s i t u a t i o n t h a t p r e v a i l s i n the r a t  resembles most c l o s e l y t h a t seen i n the golden hamster.  The  important d i f f e r e n c e , however, from the p o i n t of view of i n t r a p e r i t o n e a l t r a c e r i n t r o d u c t i o n , i s t h a t the p e r i o v a r i a n b u r s a o f the r a t has on i t s ventromedial s i d e a s m a l l opening by which the p e r i t o n e a l c a v i t y and the p e r i o v a r i a n space communicate.  Due  to t h i s d i r e c t communication  i t was  may  not  n e c e s s a r y t o s u r g i c a l l y remove or r e t r a c t the bursa i n order t o expose the o v a r i a n e p i t h e l i a l s u r f a c e more f u l l y .  Even though  t h i s procedure might have e x p e d i t e d t r a c e r movement, i t would have added unnecessary s u r g i c a l c o m p l i c a t i o n s t o the procedure. These c o m p l i c a t i o n s c o u l d be avoided by i n c r e a s i n g the time o f exposure t o t r a c e r , which would overcome the r e s t r i c t i o n on b u l k f l u i d movement which a s i n g l e s m a l l b u r s a l opening would be expected to pose. Immediately  a f t e r s e v e r i n g i t s attachments the encapsulated  ovary was removed and p l a c e d i n f i x a t i v e .  The b u r s a was  then  c a r e f u l l y d i s s e c t e d away t o promote a more r a p i d f i x a t i o n o f the germinal e p i t h e l i u m .  A f t e r p r e l i m i n a r y experiments,  v a s c u l a r p e r f u s i o n f i x a t i o n was advantage structure.  intra-  deemed to be of no added  i n p r e s e r v a t i o n of germinal e p i t h e l i a l c e l l  ultra-  L i k e a l l e p i t h e l i a , the germinal e p i t h e l i u m i s not  vascularized.  Thus i t would not be s u b j e c t to the a c t i o n of  14  the f i x a t i v e by d i f f u s i o n from b l o o d v e s s e l s any sooner than i t would be with immersion  fixation.  In those a n e s t h e t i z e d animals i n which the o v a r i e s were immersed i n f e r r i t i n s o l u t i o n , the b a s i c s u r g i c a l procedure s i m i l a r to t h a t j u s t d e s c r i b e d .  was  However, the p e r i o d of time  between exposing the o v a r i e s and s a c r i f i c i n g the animal  was  extended t o correspond to s a c r i f i c e times o f animals r e c e i v i n g intraperitoneal tracer injections.  A f t e r d i v i d i n g the d o r s a l  tendinous ligaments, adipose t i s s u e s u r r o u n d i n g each ovary i t s adnexa was  t e a s e d away.  Each u t e r i n e horn, t o g e t h e r with  i t s a t t a c h e d o v i d u c t and ovary, was  r a i s e d so t h a t the ovary  c o u l d be p l a c e d i n a s m a l l p l a s t i c c o n t a i n e r f i l l e d with s o l u t i o n (the c o n t a i n e r was  and  ferritin  intra-abdominally situated).  The  p e r i o v a r i a n b u r s a was removed i n some animals p r i o r t o t h i s step.  The s m a l l i n t e s t i n e was  r e p o s i t i o n e d w i t h i n the  c a v i t y and the c u t edges o f the i n c i s i o n approximated. the d e s i r e d p e r i o d of immersion p l a c e d by f i x a t i v e .  abdominal After  the f e r r i t i n s o l u t i o n was  dis-  A f t e r b r i e f i n s i t u f i x a t i o n the o v a r i e s  were removed and p l a c e d i n f r e s h f i x a t i v e f o r a f u r t h e r p e r i o d of  time. A f t e r removal of the o v a r i e s , a l l experiments  were terminated  by c u t t i n g the i n f e r i o r vena cava and a l l o w i n g the animals t o exsanguinate. The f i x a t i v e used i n t h i s study c o n s i s t e d of a mixture of 2% paraformaldehyde  and 2.5%  b u f f e r a d j u s t e d t o pH 7.3  g l u t a r a l d e h y d e i n 0.1  M cacodylate  ( a l i l d i l u t i o n o f the f i x a t i v e  d e s c r i b e d by Graham and Karnosvsky,  1966a).  Ovaries were  15  f i x e d f o r 3-20 h r a t room temperature (20° C ) .  After fixation,  the procedures used f o r p r o c e s s i n g t i s s u e s f o r l i g h t and t r a n s m i s s i o n e l e c t r o n microscopy d i v e r g e d p a r i n g specimens f o r scanning  from those used f o r pre-  e l e c t r o n microscopy.  F o r l i g h t and e l e c t r o n m i c r o s c o p i c  studies, fixed  ovaries  were embedded i n 7% agar, s e c t i o n e d on a S o r v a l l TC-2 t i s s u e chopper a t 200 jam,  r i n s e d s e v e r a l times i n c o l d 0.1 M  cacodylate  b u f f e r , and s t o r e d i n b u f f e r a t 4 ° C f o r 1-48 h r . Sections  from o v a r i e s which had been exposed t o HRP were  then t r a n s f e r r e d from b u f f e r t o an i n c u b a t i o n s o l u t i o n which contained  the s u b s t r a t e f o r HRP.  The s o l u t i o n c o n s i s t e d of  0.05% 3,3 -diaminobenzidine t e t r a h y d r o c h l o r i d e i n 0.05 M t r i s ,  b u f f e r c o n t a i n i n g 0.01# hydrogen peroxide Karnovsky, 1966b).  a t pH 7.6 (Graham and  The sections were exposed f o r 40 min a t  room temperature and were then a g a i n r i n s e d i n 0.1 M  cacodylate  buffer. A l l s e c t i o n s were then p o s t - f i x e d i n b u f f e r e d 1% osmium t e t r o x i d e f o r 1 h r , r i n s e d i n d i s t i l l e d water, and s t a i n e d en b l o c i n a s a t u r a t e d aqueous s o l u t i o n o f u r a n y l acetate f o r 1 hr.  T i s s u e s were then dehydrated through an ascending  e t h a n o l s e r i e s and propylene oxide, embedded i n a l i l Thick  i n f i l t r a t e d i n vacuo, and  mixture o f e p o n - a r a l d i t e  polymerized  a t 60° C.  (0.5jum) and t h i n ( s i l v e r - g r e y ) s e c t i o n s were c u t on  an LKB Ultrotorae I I I , u s i n g e i t h e r g l a s s o r diamond k n i v e s . Thick s e c t i o n s were s t a i n e d with  1% aqueous t o l u i d i n e blue and  were used f o r p r e l i m i n a r y l i g h t m i c r o s c o p i c o r i e n t a t i o n o f areas o f i n t e r e s t .  i d e n t i f i c a t i o n and  T h i n s e c t i o n s were mounted  16  on uncoated copper g r i d s and e l e c t r o n microscope.  examined i n a P h i l i p s 200  or  300  A l l t h i n s e c t i o n s were f i r s t examined  without a d d i t i o n a l membrane s t a i n i n g t o a v o i d m i s i n t e r p r e t a t i o n of r e s u l t s due  to p o s s i b l e l e a d c i t r a t e s t a i n i n g a r t e f a c t s .  In the case o f s e c t i o n s not s t a i n e d with l e a d c i t r a t e , operating voltage 40  o f the microscope was  the  reduced from 60  kV  to  kV t o enhance c o n t r a s t . F o r scanning  procedure was  e l e c t r o n microscopic  used.  s t u d i e s the f o l l o w i n g  A f t e r i n i t i a l f i x a t i o n , o v a r i e s were  r i n s e d i n s e v e r a l changes o f c o l d 0.1 A f t e r being s t o r e d i n b u f f e r at k°  M cacodylate  C f o r 1-48  buffer.  h r , o v a r i e s were  p o s t - f i x e d f o r 1 hr i n b u f f e r e d 1% osmium t e t r o x i d e . then r i n s e d i n d i s t i l l e d water and ethanol s e r i e s .  The  e t h a n o l was  dehydrated through an ascending  then s u b s t i t u t e d by  a c e t a t e by p r o c e s s i n g the t i s s u e s through an a c e t a t e s e r i e s of i n c r e a s i n g iso-amyl Iso-amyl a c e t a t e  They were  iso-amyl  ethanol-iso-amyl  acetate  concentration.  i s a p o l a r s o l v e n t m i s c i b l e with carbon d i o x i d e ,  which i s used as the t r a n s i t i o n a l f l u i d i n the c r i t i c a l d r y i n g procedure.  Carbon d i o x i d e prevents i c e c r y s t a l  d u r i n g the procedure.  Ovaries  mounted on aluminum stubs, and evaporator.  point formation  were c r i t i c a l p o i n t d r i e d , coated with g o l d i n a vacuum  Specimens were examined i n a Cambridge  Stereoscan  Model S4 microscope. C o n t r o l animals, r e p r e s e n t i n g the same age experimental scanning  animals, were a l s o prepared f o r t r a n s m i s s i o n  e l e c t r o n microscopic  described.  groups as  the and  examination by the methods j u s t  C o n t r o l animals r e c e i v e d e i t h e r one  o f two  treatments.  17 Some animals r e c e i v e d  no  to a t r a c e r i n j e c t i o n . removed and  i n t r a p e r i t o n e a l i n j e c t i o n corresponding They were a n e s t h e t i z e d and  p r o c e s s e d f o r examination.  t o show the normal s t r u c t u r e r e v e a l any  of the  their  T h i s would be  ovaries  expected  germinal e p i t h e l i u m  and  endogenous f e r r i t i n or f e r r i t i n - l i k e molecules.  As w e l l , when r e a c t e d  i n the  incubation  peroxidase a c t i v i t y would be shown. experimental r e s u l t s , any  medium, any  endogenous  By comparison w i t h  changes due  to the  the  tracer injection  by v i r t u e of e i t h e r i t s volume or i t s composition would a l s o be  revealed. The  second group of animals r e c e i v e d  an  intraperitoneal  i n j e c t i o n of i s o t o n i c s a l i n e of a volume s i m i l a r to t h a t t r a c e r a d m i n i s t e r e d t o an animal of the same weight.  of  After  a  p e r i o d c o r r e s p o n d i n g to the s a c r i f i c e time o f an animal r e c e i v i n g a t r a c e r i n j e c t i o n , the  ovaries  processed.  any  detected. could and  In t h i s way Any  then be  of the c o n t r o l animal were  changes due  other changes seen i n the g e r m i n a l a t t r i b u t e d to the  some of the  f o r endogenous peroxidase  another.  As  o v a r i a n t i s s u e was  activity.  i n the also  be  epithelium  t r a c e r i t s e l f or t o the  t r a c e r i n combination with one  c o n t r o l group,  to the s a l i n e c o u l d  saline first  reacted  I -  18  RESULTS A.  Normal Germinal E p i t h e l i u m The  f o l l o w i n g r e s u l t s apply t o a l l animals, both p r e - and  p o s t p u b e r t a l , r e c e i v i n g e i t h e r no i n t r a p e r i t o n e a l i n j e c t i o n (other than a n e s t h e t i c ) 1.  o r an i n j e c t i o n o f s a l i n e alone.  General E p i t h e l i a l Morphology As seen with the l i g h t and t r a n s m i s s i o n e l e c t r o n micro-  scopes, o v a r i a n germinal e p i t h e l i u m i n s e c t i o n ranges from a simple  squamous t o a simple  intermediate and  c u b o i d a l type, with a l l p o s s i b l e  variations ( f i g . 1).  extended by scanning  This f i n d i n g  e l e c t r o n microscopic  was  corroborated  s t u d i e s which  showed o v e r a l l s u r f a c e morphology o f the germinal e p i t h e l i a l c e l l s as w e l l as the r e g i o n a l d i s t r i b u t i o n o f c e l l shape.  This  c o r r e l a t e d with the degree o f m a t u r a t i o n o f u n d e r l y i n g f o l l i c l e s (see D i s c u s s i o n ) .  Cuboidal  or c r y p t s between f o l l i c l e s .  ;  c e l l s are found i n the " v a l l e y s " The c u b o i d a l c e l l s g i v e way i n  a graded f a s h i o n t o squamous c e l l s o v e r l y i n g the f o l l i c l e s (fig.  2).  Subsequent examination o f s e c t i o n e d m a t e r i a l showed  t h a t no c y t o l o g i c a l d i f f e r e n c e s other than shape d i s t i n g u i s h these two c e l l types.  T y p i c a l examples of c u b o i d a l and squamous  c e l l s are shown i n f i g u r e s 3 and 4. In a l l specimens examined, the germinal e p i t h e l i a l seem t o r e p r e s e n t  cells  a continuous c e l l u l a r c o v e r i n g f o r the ovary.  O c c a s i o n a l l y , i n scanning  e l e c t r o n micrographs, s i t e s o f apparent  i n d i v i d u a l c e l l n e c r o s i s are seen ( f i g . 2 ) .  In c o n t r a s t , more  19  e x t e n s i v e areas o f denudation observed  o f e p i t h e l i a l c e l l s have been  i n s t u d i e s o f the human ovary  The e p i t h e l i a l  (Papadaki and B e i l b y , 1971).  c e l l s l i e on a basement membrane of v a r i a b l e  t h i c k n e s s ( f i g s . 1,3,4).  They are s e p a r a t e d from the r e g i o n of  the d e v e l o p i n g f o l l i c l e s by the t u n i c a a l b u g i n e a o v a r i i p r e - and p o s t p u b e r t a l animals.  i n both  No c o n t i n u i t y i s observed between  the germinal e p i t h e l i a l c e l l s and the f o l l i c u l a r c e l l s of the cortex.  The basement membrane merges i m p e r c e p t i b l y with the  contents of the i n t e r c e l l u l a r  c l e f t s and with the  m a t r i x o f the t u n i c a a l b u g i n e a o v a r i i . randomly s c a t t e r e d throughout  interstitial  Collagen fibres  are  the basement membrane but seem  to be more abundant i n i t s deeper r e g i o n s ( f i g s . 1,3 »**•). 2.  C e l l Membrane Features a.  L a t e r a l c e l l membranei  intercellular  c l e f t s and  junc-  tions . The most s t r i k i n g  f e a t u r e of the i n t e r c e l l u l a r  clefts  the germinal e p i t h e l i u m i s the extreme ' v a r i a b i l i t y o f course  ( f i g s . 1,3,4).  interdigitation  This v a r i a b i l i t y  intercellular  clefts.  their  i s a f u n c t i o n o f the  o f c y t o p l a s m i c processes of i r r e g u l a r  shape from a d j a c e n t c e l l s .  of  size  and  There i s no common p a t t e r n f o r a l l  A relatively  s h o r t and d i r e c t  c l e f t is  the e x c e p t i o n , and u s u a l l y the c l e f t s are t o r t u o u s , convoluted, or even l a b y r i n t h i n e i n t h e i r complexity c e l l s may  Many of the  o v e r l a p one another f o r c o n s i d e r a b l e d i s t a n c e s .  The most obvious way adherent  ( f i g . 5).  i n which germinal e p i t h e l i a l  i s v i a the i n t e r d i g i t a t i n g  processes  c e l l s are  j u s t mentioned.  20 T h i s i s a p u r e l y mechanical  interlocking.  Such union does not  r e q u i r e the maintenance o f a constant i n t e r c e l l u l a r d i s t a n c e f o r i t s f u n c t i o n , as do some more s p e c i a l i z e d types o f j u n c t i o n s . Nonethelesss,  membranes o f t e n p a r a l l e l one another q u i t e c l o s e l y  a l o n g the path o f a c l e f t , r e g a r d l e s s o f i t s complexity.  Thus  a v e r y t o r t u o u s c l e f t may d i s p l a y a f a i r l y r e g u l a r i n t e r c e l l u l a r distance.  However, i n some c l e f t s , e s p e c i a l l y a l o n g the b a s a l  h a l f of t h e i r course, membranes are o f t e n g r e a t l y s e p a r a t e d from each other. gaps.  These r e g i o n s sometimes l o o k merely l i k e l a r g e  U s u a l l y , however, the appearance i s s u g g e s t i v e o f l a r g e  polymorphic  vacuoles e i t h e r f u s i n g with or budding o f f one or  both o f the membranes bounding the c l e f t  ( f i g s . 1,3). The  d e n s i t y o f the contents of these " v a c u o l e s " i s i d e n t i c a l t o t h a t o f the i n t e r c e l l u l a r substance r e g i o n s o f the c l e f t s . s e c t i o n s through  o f the more c l o s e l y approximated  T h e r e f o r e , these areas probably  represent  l a r g e spaces which e x i s t due t o i r r e g u l a r i t i e s  i n the shape and p a t t e r n o f i n t e r l o c k i n g c y t o p l a s m i c  processes.  P i n o c y t o t i c i n v a g i n a t i o n s and v e s i c l e s appear q u i t e o f t e n along the course o f the membranes bounding the i n t e r c e l l u l a r clefts.  These w i l l be d i s c u s s e d f u r t h e r i n s e c t i o n 4, under  Organellar s t r u c t u r e . Small punctate approximation,  r e g i o n s or f o c i o f very c l o s e membrane  and i n some cases f u s i o n , are f r e q u e n t l y seen  along some o f the c l e f t s suggests  ( f i g . 6).  T h e i r appearance i n s e c t i o n  t h a t they may r e p r e s e n t macular forms o f gap j u n c t i o n s  or o c c l u d i n g j u n c t i o n s , o r s e c t i o n s o f z o n u l a r j u n c t i o n s o f the same t y p e s .  O f t e n there i s an i n c r e a s e i n the c y t o p l a s m i c  21  d e n s i t y subjacent  to t h i s f o c a l type o f j u n c t i o n .  There  o f t e n m u l t i p l e randomly s c a t t e r e d f o c i along a c l e f t ,  their  numbers u s u a l l y being g r e a t e r near the p e r i t o n e a l end. numerous are extended v e r s i o n s of the f o c a l j u n c t i o n s d e s c r i b e d , which s i m i l a r l y may cleft  ( f i g . ?).  whether adjacent fused.  Again there  are  Less just  be m u l t i p l e a l o n g a s i n g l e  is a difficulty  i n ascertaining  membranes are j u s t extremely c l o s e or a c t u a l l y  O c c a s i o n a l l y , l o n g r e g i o n s o f apparent membrane f u s i o n  are seen ( f i g . 8 ) .  These are s i m i l a r to those observed i n meso-  thelium by Gotran and Karnovsky (1968).  However, even these  have sometimes been r e s o l v e d to a c t u a l l y be composed o f d i s t i n c t c e l l membranes separated  by a s m a l l but  two  definite  i n t e r c e l l u l a r space ( f i g . 9 ) . b.  A p i c a l c e l l membrane.  C e l l a p i c a l s u r f a c e m o d i f i c a t i o n s appear mainly as microvilli  and p i n o c y t o t i c v e s i c l e s and  invaginations.  The  numbers  and d i s t r i b u t i o n of m i c r o v i l l i are q u i t e random from c e l l c e l l and  are b e s t a p p r e c i a t e d with scanning  of the c e l l f r e e s u r f a c e  ( f i g . 10).  e l e c t r o n micrographs  In s e c t i o n the  microvilli  are seen t o c o n t a i n ground cytoplasm and an i n t e r n a l of microfilaments  p a r a l l e l to the l o n g a x i s .  to  skeleton  Pinocytotic  i n v a g i n a t i o n s are seen to open on to the f r e e s u r f a c e i n both t r a n s m i s s i o n and scanning The  e l e c t r o n micrographs ( f i g s .  s i z e o f the i n v a g i n a t i o n s i s v a r i a b l e , as i s t h e i r  bution.  I r r e g u l a r cytoplasmic  as d e s c r i b e d i n other s p e c i e s 1971), are sometimes seen.  evaginations (Gondos, 1969;  3i^ill)« distri-  of the f r e e s u r f a c e , Papadaki and  Beilby,  C i l i a are seen only i n f r e q u e n t l y ,  22 even i n s c a n n i n g e l e c t r o n m i c r o s c o p i c s t u d i e s , c.  B a s a l c e l l membrane.  The contour o f the c e l l b a s a l s u r f a c e v a r i e s from to  c e l l , b e i n g r e l a t i v e l y s t r a i g h t i n some and h i g h l y  i n others.  cell irregular  P i n o c y t o t i c v e s i c l e s open on t o t h i s s u r f a c e , although  they are fewer  i n number than a t the f r e e s u r f a c e .  There are  no j u n c t i o n a l s p e c i a l i z a t i o n s a l o n g the b a s a l membrane. 3.  Nuclear S t r u c t u r e Germinal e p i t h e l i a l c e l l n u c l e a r shape r e f l e c t s t h a t o f the  cell  (figs.  3,4).  N u c l e i have o v e r a l l shapes i n s e c t i o n r a n g i n g  from f l a t and e l o n g a t e d t o almost c i r c u l a r .  The r e l a t i v e  p r o p o r t i o n o f the c e l l o c c u p i e d by the nucleus v a r i e s w i t h the plane o f s e c t i o n . (figs.  1,4).  The n u c l e a r contour i s u s u a l l y  Cytoplasmic p r o j e c t i o n s o f v a r y i n g dimensions  extend i n t o the nucleus ( f i g . to  1), and sometimes cause the nucleus  assume a p s e u d o l o b u l a t e d form.  M i t o c h o n d r i a and v e s i c l e s •  are sometimes seen i n these e x t e n s i o n s . a t e d by the u s u a l b i l a m e H a t e n u c l e a r pores are found. t h i n diaphragm. the envelope  irregular  The nucleus i s d e l i n e -  n u c l e a r envelope, a l o n g which  Some o f these pores are c l o s e d by a  Ribosomes are adherent t o the outer l a m e l l a o f  (fig.  ? ) . Most o f the chromatin i s c i r c u m f e r e n t i a l l y  d i s t r i b u t e d a g a i n s t the i n n e r aspect o f the n u c l e a r envelope, w i t h some l o c a t e d as coarse clumps throughout the nucleus ( f i g s . 1,3,4).  A l l n u c l e i examined were i n the i n t e r p h a s e s t a t e .  N u c l e o l i are r a r e l y c u t i n s e c t i o n ( f i g . 4).  23  4.  Organellar Structure Rough endoplasmic r e t i c u l u m i s found i n v a r i a b l e amounts  throughout the germinal e p i t h e l i a l c e l l cytoplasm.  It is  u s u a l l y c l o s e l y a s s o c i a t e d with m i t o c h o n d r i a , with which i t may form l a r g e aggregates ( f i g s . 1,3,4,7).  The contents o f the  c i s t e r n a e appear to be o f about the same d e n s i t y as the c y t o plasm.  The degree of d i l a t i o n o f the c i s t e r n a e v a r i e s both  w i t h i n a c e l l and from c e l l t o c e l l .  Smooth endoplasmic  r e t i c u l u m i s v e r y seldom seen. Both the m i t o c h o n d r i a r e l a t e d t o the rough endoplasmic i • r e t i c u l u m and those d i s t r i b u t e d randomly throughout the c y t o plasm have a s i m i l a r appearance.  The matrix i s denser than the  s u r r o u n d i n g cytoplasm and the c o n t e n t s o f most v e s i c l e s ,  and  i n s e c t i o n m i t o c h o n d r i a l shape v a r i e s from c i r c u l a r t o o v o i d to  elongate ( f i g s . 3,4).  C r i s t a e appear as f l a t t e n e d  lamellae  whose o r i e n t a t i o n w i t h i n the m i t o c h o n d r i a i s not s p e c i f i c . The G o l g i complex c o n s i s t s o f a v a r i a b l e number o f l a m e l l a e of  d i f f e r e n t shape and s i z e .  observed i n each c e l l .  U s u a l l y only one G o l g i complex i s  I t i s situated i n a l a t e r a l or superior  p a r a n u c l e a r p o s i t i o n i n most i n s t a n c e s .  I t i s not observed  between the nucleus and the b a s a l c e l l s u r f a c e . presumably d e r i v e d from the G o l g i complex  Vesicles  are seen about i t s  e n t i r e p e r i m e t e r and are n o t p r e f e r e n t i a l l y r e l a t e d t o any p a r t i c u l a r a s p e c t o f the complex  (fig. 4 inset).  ,  The degree o f  f i l l i n g and e l e c t r o n d e n s i t y of the contents o f these v e s i c l e s is  variable. Ribosomes are v e r y numerous.  As w e l l as b e i n g c l o s e l y  24  a s s o c i a t e d with the endoplasmic  r e t i c u l u m and n u c l e a r envelope,  they are observed i n l a r g e numbers f r e e i n the cytoplasm and occasionally  i n p o l y r i b o s o m a l groupings.  Cytoplasmic v e s i c l e s are seen throughout the c e l l and a t a l l c e l l surfaces.  They are o f h i g h l y d i v e r s e s i z e and shape.  Many o f the v e s i c l e s are near the a p i c a l and l a t e r a l c e l l s u r f a c e s and may be d e r i v e d from p i n o c y t o t i c a c t i v i t y . a l s o be p i n o c y t o t i c  They c o u l d  i n v a g i n a t i o n s s e c t i o n e d i n a plane other  than t h a t i n which t h e i r opening t o a s u r f a c e can be seen. The amount and d i s t r i b u t i o n o f e l e c t r o n dense m a t e r i a l w i t h i n a v e s i c l e ranges c o n s i d e r a b l y , as p r e v i o u s l y noted f o r G o l g i vesicles.  Some v e s i c l e s c o n t a i n what appear t o be s m a l l e r  v e s i c l e s i n s i d e them, thus forming m u l t i v e s i c u l a r bodies ( f i g . 4 inset).  Somewhat denser s t r u c t u r e s s i m i l a r t o these have been  i d e n t i f i e d as autophagic vacuoles i n human germinal e p i t h e l i u m (Papadaki and B e i l b y , 1971). Cytoplasmic i n c l u s i o n s which may r e p r e s e n t l i p i d s t o r e s are seen i n some c e l l s  ( f i g s . 3,4).  T h e i r s i z e and d i s t r i b u t i o n  w i t h i n a germinal e p i t h e l i a l c e l l i s n o t constant and i n s e c t i o n u s u a l l y no more than two or t h r e e are ever noted w i t h i n a c e l l . The s u b j a c e n t s t r o m a l and g r a n u l o s a c e l l s u s u a l l y c o n t a i n more l i p i d i n c l u s i o n s than the e p i t h e l i a l  cells.  M i c r o f i l a m e n t s are seen i n the cytoplasm o f some germinal e p i t h e l i a l c e l l s ; u s u a l l y as bundles with no p a r t i c u l a r orientation.  25  B.  Tracer Experiments In a l l animals exposed to tracer, either HRP  or f e r r i t i n ,  no departure from the normal ultrastrueture of the germinal epithelium or subjacent ovarian tissue i s seen.  The only  difference noted i s the presence of tracers within these tissues. Although both HRP  and f e r r i t i n appear i n the ovary within 45 min  of exposure to tracer, they d i f f e r markedly i n t h e i r depth of penetration.  HRP  i s found not only i n the germinal epithelium  but also deep within the tunica albuginea o v a r i i within t h i s period of time.  F e r r i t i n on the other hand appears only i n  germinal e p i t h e l i a l c e l l s at the end of 45 min, and requires several hours before i t appears i n the c e l l s and  interstitium  of the tunica albuginea o v a r i i . Endogenous peroxidase a c t i v i t y i s limited to the cytoplasm of erythrocytes within ovarian blood vessels.  No electron dense  p a r t i c l e s resembling f e r r i t i n are seen. 1.  HRP  Distribution  HRP  i t s e l f can not actually be seen very well i n an electron  micrograph.  Rather i t i s the electron dense enzymatic reaction  product which i s v i s u a l i z e d as the tracer.  Adhering to con-  vention, however, the reaction product w i l l be referred to as HRP.v  The v i s i b i l i t y of the reaction product i s further enhanced  a f t e r i t s reaction with osmium tetroxide during post-fixation (Graham and Karnovsky, 1966b). HRP  i s observed on the germinal e p i t h e l i a l c e l l a p i c a l  surface, i n the i n t e r c e l l u l a r c l e f t s , i n pinocytotic invagina-  26  t i o n s , a t a l l e p i t h e l i a l (and some f i b r o b l a s t ) c e l l  surfaces,  i n v e s i c l e s and m u l t i v e s i c u l a r bodies o f e p i t h e l i a l c e l l s and fibroblasts,  i n the basement membrane, and i n the i n t e r s t i t i u m  o f the t u n i c a a l b u g i n e a  ovarii  ( f i g . 12).  The p a t t e r n of t r a c e r  uptake was s i m i l a r i n a l l experiments. HRP i s observed t o be most abundant i n the e x t r a c e l l u l a r regions  o f the ovary.  There i s a marked e x t r a c e l l u l a r g r a d i e n t  o f HRP d e n s i t y from the e p i t h e l i a l f r e e s u r f a c e through the i n t e r c e l l u l a r c l e f t s , basement membrane, and i n t e r s t i t i u m ( f i g . 13).  The e x t e n t o f the g r a d i e n t v a r i e s with the time o f exposure  to HRP.  C o l l a g e n f i b r e s are e s p e c i a l l y w e l l v i s u a l i z e d i n  the basement membrane and i n t e r s t i t i u m due t o the negative ing  stain-  e f f e c t o f HRP. P i n o c y t o t i c i n v a g i n a t i o n s c o n t a i n i n g v a r i a b l e amounts o f  HRP are seen a t a l l s u r f a c e s o f the germinal e p i t h e l i a l p a r t i c u l a r l y the a p i c a l and l a t e r a l s u r f a c e s .  Such  cell,  invagina-  t i o n s are a l s o seen, b u t much l e s s f r e q u e n t l y , a t the e p i t h e l i a l c e l l b a s a l s u r f a c e and a t the s u r f a c e of f i b r o b l a s t s .  Occasion-  a l l y HRP was not seen a t the a p i c a l s u r f a c e , even though i t appeared elsewhere throughout the e p i t h e l i u m and subjacent t i s s u e s ( f i g . 14). T h i s l o c a l removal o f HRP was a t t r i b u t a b l e to excessive  probably  washing of t i s s u e i n b u f f e r p r i o r t o  p o s t - f i x a t i o n , and has been noted i n other studies:where HRP has  been used as a t r a c e r (Cotran and Karnovsky, 1968). V e s i c l e s c o n t a i n i n g HRP are found throughout germinal  epithelial cells.  They c o n s t i t u t e a v a r i a b l e but reasonably  s m a l l p r o p o r t i o n of the t o t a l number of c e l l v e s i c l e s . Even a f t e r exposure to HRP  f o r 5 h r s , the p r o p o r t i o n of t r a c e r -  c o n t a i n i n g v e s i c l e s was are completely  s t i l l quite small.  Some v e s i c l e s  f i l l e d with the t r a c e r , which appears e i t h e r to  be amorphous or to have a coarse g r a n u l a r appearance. v e s i c l e s which are not completely  f i l l e d with HRP,  In  other  the t r a c e r  appears as s m a l l clumps s i m i l a r to those o f t e n seen at the a p i c a l s u r f a c e , or as an i n n e r c i r c u m f e r e n t i a l c o a t i n g . c o n t a i n i n g HRP but not  cell  Vesicles  are sometimes seen i n f i b r o b l a s t s ( f i g . 12),  i n capillary endothelial c e l l s .  P e r i p h e r a l l y , v e s i c l e s are more numerous near the a p i c a l and  l a t e r a l c e l l s u r f a c e s than the b a s a l s u r f a c e .  s u r f a c e v e s i c l e s are of a f a i r l y  uniform  as are those at the b a s a l s u r f a c e . c e l l s u r f a c e may  contour,  V e s i c l e s near the  lateral  e i t h e r be s i m i l a r to those seen a t the  c e l l s u r f a c e s , or they may The  s i z e and  Apical  other  be q u i t e v a r i a b l e i n s i z e and  d i s t r i b u t i o n of v e s i c l e s a t the a p i c a l and  lateral  shape.  surfaces  p a r a l l e l s t h a t of the p i n o c y t o t i c i n v a g i n a t i o n s d e s c r i b e d After excessive  earlier.  b u f f e r washing the p r o p o r t i o n o f a p i c a l s u r f a c e  v e s i c l e s c o n t a i n i n g HRP  i s g r e a t l y reduced ( f i g . 14).  This  would not be expected o f t r u e v e s i c l e s and suggests t h a t many s o - c a l l e d " v e s i c l e s " devoid o f t r a c e r subsequent to  excessive  washing are i n f a c t p i n o c y t o t i c i n v a g i n a t i o n s continuous the c e l l a p i c a l s u r f a c e i n another plane.  with  There are, however,  other t r a c e r - c o n t a i n i n g v e s i c l e s w i t h i n the c e l l s whose are not removed by prolonged washing ( f i g . 12). v e s i c l e s some are q u i t e c l o s e to the l a t e r a l c e l l  contents  Of these surfaces  28  bounding the i n t e r c e l l u l a r c l e f t s .  C e r t a i n of these c o u l d  be  true v e s i c l e s d e r i v e d from the l a t e r a l c e l l s u r f a c e , whereas others  c o u l d be  i n v a g i n a t i o n s s i m i l a r to those seen a t  a p i c a l surface.  the  In e i t h e r case they c o u l d have been f i l l e d  with t r a c e r by d i f f u s i o n from the i n t e r c e l l u l a r c l e f t s .  The  p o s s i b i l i t y e x i s t s t h a t they c o u l d a l s o be v e s i c l e s from  the  a p i c a l s u r f a c e emptying t h e i r contents  i n t o the  intercellular  clefts. As e a r l i e r observed, the i r r e g u l a r i n t e r l o c k i n g o f c y t o plasmic  processes  from adjacent  e p i t h e l i a l c e l l s often gives  r i s e t o the appearance of l a r g e i n v a g i n a t i o n s i n t o the from the l a t e r a l c e l l s u r f a c e s .  cell  S e c t i o n i n g through such  an  i n v a g i n a t i o n i n a plane other than t h a t i n which i t i s continuous with the membrane of the i n t e r c e l l u l a r c l e f t may  cause  the  a r t e f a c t u a l appearance of t r a c e r - f i l l e d v e s i c l e s of v a r i a b l e contour.  Some of these "vesicles"may  be q u i t e removed from  the l a t e r a l c e l l s u r f a c e , depending on the l e n g t h of invagination. very l o n g .  As seen i n f i g . 15t  the  these i n v a g i n a t i o n s can  In t h i s p a r t i c u l a r case,  be  s e c t i o n i n g i n another plane  c o u l d e a s i l y r e s u l t i n the c r e a t i o n of an apparent v e s i c l e deep w i t h i n the c e l l .  On o c c a s i o n the appearance i s a l s o c r e a t e d  of  l a r g e t r a c e r - f i l l e d v e s i c l e s to which are connected s m a l l e r vesicles,  These s m a l l e r v e s i c l e s seem to be e i t h e r forming  f r o m , o r emptying t h e i r contents 15  inset).  i n t o , the l a r g e v e s i c l e ( f i g .  However, such l a r g e v e s i c l e s are always s i t u a t e d  near i n t e r c e l l u l a r c l e f t s , never deep w i t h i n a c e l l . are a l s o seen i n c o n t r o l m a t e r i a l .  They  29  There s t i l l remain a number o f t r a c e r - f i l l e d v e s i c l e s w i t h i n the c e l l which are so s i t u a t e d t h a t i t would be to envisage t h e i r c o n n e c t i o n  with any  of the c e l l  difficult  surfaces.  They are on average s m a l l e r and more r e g u l a r i n o u t l i n e than would be expected of "vesicles'* d e r i v e d from s e c t i o n of l a t e r a l surface invaginations. existence  One  would have to invoke the  of very l o n g and numerous s u r f a c e membrane  invagina-  t i o n s of s m a l l and r e g u l a r diameter p e n e t r a t i n g deeply  i n t o the  c e l l which, when c u t i n c r o s s - s e c t i o n , would appear as v e s i c l e s . I f t h i s were the case,  then o b l i q u e and  longitudinal sections  o f such channels should be seen i n some micrographs, and are not.  To completely  s e c t i o n s were examined. these v e s i c l e s from any  eliminate t h i s p o s s i b i l i t y  they  serial  This demonstrated the independence o f c e l l surface.  There are thus some t r a c e r -  f i l l e d v e s i c l e s which would seem t o c o n s t i t u t e d e f i n i t e evidence of the v e s i c u l a r uptake of HRP.  Few  v e s i c l e s are seen to open  onto the basement membrane, s u g g e s t i n g does occur,  that i f tracer transport  i t i s e i t h e r not i n the d i r e c t i o n of the  s u r f a c e or occurs  to only a minor extent  basal  i n that d i r e c t i o n .  It  i s not p o s s i b l e to t e l l whether or not such v e s i c l e s a c t i v e l y t r a n s p o r t t r a c e r from the c e l l a p i c a l s u r f a c e or to or from the cell  lateral  surfaces.  Within the  i n t e r c e l l u l a r c l e f t s HRP  homogeneous ( f i g s . 16,1?), or i t may ance ( f i g . 18).  The  may  present  be  reasonably  a granular  presence o f the t r a c e r w i t h i n the  emphasizes t h e i r t o r t u o s i t y .  The  c o n t i n u i t y of HRP  f r e e s u r f a c e to the basement membrane v a r i e s w i t h i n  appearclefts  from the different  cell  30  clefts. o f the  I t seems to be a f u n c t i o n of the presence o f types mentioned e a r l i e r (compare f i g s ,  Unfortunately,  the,presence of HRP  junctions  16,1?,18).  w i t h i n a c l e f t o f t e n seems  t o obscure the l a t e r a l membrane s u r f a c e s .  Thus j u n c t i o n a l  s p e c i a l i z a t i o n s may  be d i f f i c u l t to d i s c e r n and i n t e r p r e t  a l o n g such c l e f t s .  In some c l e f t s where d e f i n i t e f o c a l  or extended v e r s i o n s  t h e r e o f can be seen, HRP  junctions  seems t o be  found  on e i t h e r s i d e of the p o i n t of c l o s e c e l l approximation. cannot be d i s t i n g u i s h e d whether HRP junctional area  i s present  i t s e l f or whether i t has  within  the  d i f f u s e d around  j u n c t i o n to appear on both s i d e s of i t ( f i g . 17). or more f o c a l j u n c t i o n s are seen a l o n g a c l e f t , the between adjacent  It  j u n c t i o n s i s sometimes devoid  When  the two  region  of t r a c e r ( f i g , 18).  This suggests t h a t some j u n c t i o n s are d e f i n i t e l y not permeable to HRP.  I t a l s o suggests t h a t these p a r t i c u l a r j u n c t i o n s  not simple maculae occludentes, d i f f u s e d around them to f i l l j u n c t i o n s may  otherwise HRP  These  c r o s s s e c t i o n s through  annular j u n c t i o n between adjacent  an  l a t e r a l c e l l surfaces,  a l o c a l r i n g of o c c l u s i o n would prevent HRP i n i t s i n t e r i o r and  c o u l d have  the i n t e r v e n i n g space,  perhaps r e p r e s e n t  are  from being  Such  localized  would l e a d to the appearance o f a r e g i o n  such as shown i n f i g . 18.  Again, f r e e z e - f r a c t u r e s t u d i e s would  probably be the most s u i t a b l e method f o r p o s i t i v e l y e s t a b l i s h i n g the nature o f the 2.  j u n c t i o n s of the germinal  epithelium.  Ferritin Distribution F e r r i t i n l o c a l i z a t i o n i n o v a r i e s o f animals i n j e c t e d with  31  f e r r i t i n and in f e r r i t i n  i n animals whose o v a r i e s were d i r e c t l y i s essentially similar.  e x t r a c e l l u l a r l y , with the notable  immersed  I t i s found i n t r a -  exception  and  t h a t i t i s not  found i n the i n t e r c e l l u l a r c l e f t s of the g e r m i n a l  epithelium.  Subsequent to f e r r i t i n exposure, a v a r i a b l e number of germinal e p i t h e l i a l c e l l s are seen t o c o n t a i n f e r r i t i n  within  iriembrane-bounded v e s i c l e s and m u l t i v e s i c u l a r bodies ( f i g . 19) • F e r r i t i n molecules are not u s u a l l y observed i n e p i t h e l i a l intercellular clefts  (see D i s c u s s i o n ) .  the t u n i c a a l b u g i n e a  ( f i g . 20)  c e l l s ( f i g . 21)  Some f i b r o b l a s t s o f  and some c a p i l l a r y e n d o t h e l i a l  also contain f e r r i t i n within v e s i c l e s .  p o s s i b l e sequence of the p i n o c y t o t i c events u n d e r l y i n g uptake i s shown i n f i g . 22. containing f e r r i t i n  A ferritin  I t i s r e a l i z e d t h a t the v e s i c l e  i n f i g . 22 c o u l d a c t u a l l y be an  invagination  s e c t i o n e d i n a plane i n which i t i s not connected t o the Nevertheless, and  as f e r r i t i n  i s probably  surface.  i s found deep to the germinal e p i t h e l i u m  not t r a n s p o r t e d through the i n t e r c e l l u l a r  clefts  (see D i s c u s s i o n ) , i t i s assumed t h a t the sequence shown i n fig.  22 a c t u a l l y m i r r o r s the i n i t i a l events i n v e s i c u l a r uptake  of f e r r i t i n .  O c c a s i o n a l l y , e p i t h e l i a l c e l l s and f i b r o b l a s t s  appear t o c o n t a i n f r e e f e r r i t i n molecules i n the cytoplasm. F e r r i t i n i s sometimes seen i n granulosa the zona p e l l u c i d a or i n oocytes. and venules o f the t u n i c a a l b u g i n e a fluid  ( f i g , 23)  c e l l v e s i c l e s , but not i n  Blood plasma w i t h i n ( f i g . 21)  and  capillaries  follicular  are f r e q u e n t l y seen to c o n t a i n f r e e  ferritin  molecules.  F e r r i t i n molecules are always seen t o be d i s c r e t e  particles.  They do not form the type o f amorphous clumps  32  characteristic Both the  of  HRP.  number and  d i s t r i b u t i o n of  v e s i c l e s w i t h i n a c e l l and  ferritin-containing  the number arid d i s t r i b u t i o n  of  f e r r i t i n molecules w i t h i n a s i n g l e v e s i c l e e x h i b i t wide v a r i a tion.  In s p i t e of t h e i r v a r i a b l e  numbers, f e r r i t i n - c o n t a i n i n g  v e s i c l e s r a r e l y r e p r e s e n t more than a r e l a t i v e l y s m a l l proport i o n of a l l c e l l v e s i c l e s .  The  number o f f e r r i t i n  w i t h i n a v e s i c l e ranges from as few thousand, and  both extremes may  as one  to as many as  of the  tunica  material.  There i s not  s i z e of a v e s i c l e and it.  as f r e q u e n t l y observed i n  the  large  directly.  are  larger  not  On average,  bounding the  This could  experiments, f e r r i t i n  intercellular clefts.  not  empty i n t o the  from the  as  c e l l a p i c a l surface  or i n v a g i n a t i o n s a s s o c i a t e d with the  argument t h a t v e s i c l e s  the  ferritin-containing  of s m a l l e r v e s i c l e s ,  seen to open to the  the  is  not  membranes  T h i s would seem to support a p i c a l s u r f a c e probably  do  clefts.  Even though l a r g e v e s i c l e s were not i n t o the  control  than other c e l l v e s i c l e s .  In c o n t r a s t to the HRP  found i n v e s i c l e s  of  dimensions c o n t a i n s fewer molecules  t h a t they a r i s e from f u s i o n  vesicles  Vesicles  number of f e r r i t i n molecules w i t h i n  than a much s m a l l e r v e s i c l e .  indicate  fibro-  always a d i r e c t c o r r e l a t i o n between  O f t e n a v e s i c l e of l a r g e  v e s i c l e s seem t o be  Vesicles  found i n the  albuginea o v a r i i ( f i g . 20).  comparable s i z e were not  several  occur i n a s i n g l e c e l l .  with the h i g h e s t f e r r i t i n c o n c e n t r a t i o n are blasts  molecules  basement membrane, the  deeper r e g i o n s of the  ovary and  o f t e n seen to empty  appearance of f e r r i t i n  in  i t s v i r t u a l absence i n  the  i n t e r c e l l u l a r c l e f t s would seem to imply t h a t v e s i c u l a r  the  transport  33  i s the method by which f e r r i t i n leaves the germinal  epithelial  cells. The  d i s t r i b u t i o n o f f r e e f e r r i t i n molecules i n the basement  membrane,  i n t e r s t i t i u m , blood plasma, and f o l l i c u l a r  seems t o be random. are e v i d e n t .  fluid  No p r e f e r r e d routes o f t r a c e r movement  F e r r i t i n i s e x t r a c e l l u l a r l y much l e s s abundant  than HRP and, because of i t s sparseness,  does not assume an  obvious g r a d i e n t o f d i s t r i b u t i o n l i k e t h a t of HRP. much more f e r r i t i n i s c o n t a i n e d  Even though  i n t r a c e l l u l a r l y within vesicles,  no c e l l u l a r g r a d i e n t o f t r a c e r d i s t r i b u t i o n i s e v i d e n t . due  t o v e s i c l e v a r i a t i o n i n number, s i z e , and f e r r i t i n  both w i t h i n a c e l l and from c e l l t o c e l l . to which f e r r i t i n penetrates  This i s content  However, the depth  the ovary i n c r e a s e s as time of  exposure t o f e r r i t i n i n c r e a s e s .  T h i s i s i n d i c a t e d by i t s presence  w i t h i n f i b r o b l a s t v e s i c l e s o f s u c c e s s i v e l y deeper r e g i o n s o f the tunica albuginea developing  ( f i g . 20) and w i t h i n the f o l l i c u l a r  f o l l i c l e s o f the o u t e r c o r t e x ( f i g . 23).  p e n e t r a t i o n thus seems t o be a time-dependent  f l u i d of Ferritin  process.  34  C.  Figures  Figure 1«  Overview o f germinal  e p i t h e l i u m and p a r t o f under-  l y i n g t u n i c a albuginea o v a r i i . t h e l i a l c e l l i s i n d i c a t e d (GE).  One germinal  Note the i r r e g u l a r  n u c l e a r contour  i n these c e l l s , the complex  cellular clefts  (arrows),  ment membrane of a c a p i l l a r y seen.  epi-  inter-  and the u n d e r l y i n g base-  of v a r i a b l e t h i c k n e s s .  The presence  (C) very near t o the s u r f a c e i s a l s o  S c a l e bar i n t h i s and a l l succeeding  graphs i n d i c a t e s 1 /xm.  micro-  Uranyl acetate and l e a d  c i t r a t e s t a i n i n g . X 5.800.  F i g u r e 2J  Scanning e l e c t r o n micrograph o f the o v a r i a n e p i t h e l i a l s u r f a c e showing the r e g i o n a l d i s t r i b u t i o n o f c e l l shape.  S i t e s o f apparent  c e l l n e c r o s i s are i n d i c a t e d (arrows). known whether such s i t e s occur normally germinal X 420.  individual  I t i s not i n the  e p i t h e l i u m or whether they are a r t e f a c t u a l .  Figure  3»  Typical cuboidal  germinal e p i t h e l i a l c e l l s .  Cell  on l e f t , which appears t o be detached from r e s t of epithelium lipid  due t o plane o f s e c t i o n i n g ,  inclusions (asterisks).  reticulum,  contains  Rough endoplasmic  ribosomes, and m i t o c h o n d r i a a r e abundant.  M i c r o v i l l i are a l s o numerous.  Note random arrange-  ment of c o l l a g e n f i b e r s i n basement membrane (BM) and  h i g h l y v a r i a b l e course and appearance o f i n t e r -  cellular clefts. staining.  Figure  4i  Uranyl a c e t a t e and l e a d c i t r a t e  X 10,600.  T y p i c a l squamous g e r m i n a l e p i t h e l i a l c e l l same o r g a n e l l e s nucleolus  as c u b o i d a l  c e l l i n f i g . 3.  (N) i s a l s o p r e s e n t .  lead c i t r a t e staining.  containing A  U r a n y l a c e t a t e and  X 10,600.  Inset shows G o l g i complex (G), mitochondria, and numerous i n t r a c e l l u l a r v e s i c l e s o f v a r i a b l e content and  electron density.  i s a l s o seen. staining.  A m u l t i v e s i c u l a r body (arrow)  U r a n y l a c e t a t e and l e a d c i t r a t e  X 19,000.  F i g u r e 5»  Germinal e p i t h e l i u m . complexity faces.  Intercellular cleft,  showing  o f i n t e r d i g i t a t i o n o f adjacent c e l l  sur-  Uranyl acetate and l e a d c i t r a t e s t a i n i n g .  X 37.800. F i g u r e 6«  Germinal e p i t h e l i u m .  Small punctate  junctions,  which may be gap j u n c t i o n s or macular t i g h t j u n c t i o n s , are i n d i c a t e d (arrows). citrate staining. F i g u r e 7*  Uranyl a c e t a t e and l e a d  X 56,400.  Germinal e p i t h e l i u m .  Extended r e g i o n s o f c e l l  approximation a r e shown (arrows). gap  junctions.  staining. F i g u r e 8i  These may be  Uranyl a c e t a t e and l e a d c i t r a t e  X 32,400.  Germinal e p i t h e l i u m .  Long r e g i o n o f apparent  membrane f u s i o n , c r e a t i n g appearance o f a pentala mi na r  junction.  staining. F i g u r e 9»  U r a n y l a c e t a t e and l e a d c i t r a t e  X 65,200.  Germinal e p i t h e l i u m .  D e t a i l e d s t r u c t u r e o f a junc-  t i o n which appeared t o be pentalaminar a t a lower magnification. separate  the two e x t e r n a l l e a f l e t s o f apposing  c e l l membranes. staining.  A d e f i n i t e space c a n be seen t o  Uranyl acetate and l e a d c i t r a t e  X 235.000.  40  Figure  10i  Scanning  e l e c t r o n micrograph  epithelium,  showing  of m i c r o v i l l i .  Figure  11i  Scanning  t h e numbers a n d d i s t r i b u t i o n  3,000.  e l e c t r o n micrograph  invaginations surface.  X  o f the germinal  showing  pinocytotic  of a germinal e p i t h e l i a l  Microvilli  c a n a l s o be s e e n .  cell  apical  X 15,000.  F i g u r e 121  O v e r a l l d i s t r i b u t i o n o f HRP.  Note i t s presence on  a p i c a l surface, i n i n t e r c e l l u l a r c l e f t s , i n pinocytotic  i n v a g i n a t i o n s and v e s i c l e s o f germinal  epithelial cells  (GE) and f i b r o b l a s t s  (F), i n  basement membrane, and i n i n t e r s t i t i u m o f t u n i c a albuginea o v a r i i . staining.  F i g u r e IJt  Uranyl acetate and l e a d c i t r a t e  X 5,800.  E x t r a c e l l u l a r g r a d i e n t of HRP. decreases from germinal  Density of HRP  e p i t h e l i a l c e l l (GE)  a p i c a l s u r f a c e through i n t e r c e l l u l a r  elefts,  basement membrane, and i n t e r s t i t i u m .  Uranyl  a c e t a t e and l e a d c i t r a t e s t a i n i n g .  X 16,600.  I n s e t shows i n t e r c e l l u l a r c l e f t s (arrows) o f a c o n t r o l specimen, s i m i l a r l y s t a i n e d with  uranyl  a c e t a t e and l e a d c i t r a t e , f o r purposes o f comparison re. e l e c t r o n density of c l e f t s .  X 14,300.  Figure 14i  Micrograph i l l u s t r a t e s removal o f HRP from germinal e p i t h e l i a l c e l l a p i c a l surface a f t e r excessive washing.  buffer  Note t h a t HRP i s s t i l l found i n deeper  r e g i o n s o f germinal e p i t h e l i u m and subjacent t i s s u e s . Uranyl acetate and l e a d c i t r a t e s t a i n i n g .  Figure 15»  Germinal e p i t h e l i u m . intercellular cleft  Note extended (asterisk).  X 14,500.  invagination of  Sectioning i n  another plane c o u l d r e s u l t i n c r e a t i o n o f an apparent HRP-containing v e s i c l e deep w i t h i n c e l l . a c e t a t e and l e a d c i t r a t e s t a i n i n g . Inset shows l a r g e "vesicle*"  Uranyl  X 24,300.  (arrow) near  inter-  c e l l u l a r c l e f t t o which are connected s m a l l e r v e s i c l e s Uranyl acetate and l e a d c i t r a t e s t a i n i n g .  Figure 16»  X 19,400.  I n t e r c e l l u l a r c l e f t showing c o n t i n u i t y o f HRP from p e r i t o n e a l s u r f a c e above t o basement membrane below.  Tracer-containing vesicular p r o f i l e s  (arrows) may a c t u a l l y be p i n o c y t o t i c i n v a g i n a t i o n s s i m i l a r t o those seen a t the a p i c a l and b a s a l s u r f a c e s o f the g e r m i n a l e p i t h e l i a l  cells.  Uranyl a c e t a t e and l e a d c i t r a t e s t a i n i n g .  X 73,200.  F i g u r e 17i  Germinal e p i t h e l i u m .  Intercellular cleft  showing  p o s s i b l e r e g i o n o f membrane f u s i o n (arrow) near a p i c a l end. areas.  Note the d e p o s i t i o n o f HRP i n adjacent  Uranyl a c e t a t e and l e a d c i t r a t e s t a i n i n g .  X 73,200.  F i g u r e 18i  I n t e r c e l l u l a r c l e f t showing r e g i o n o f e x c l u s i o n o f HRP (arrow), s u g g e s t i n g  t h a t some j u n c t i o n s a r e n o t  permeable t o t h i s t r a c e r .  V e s i c l e (V) i n lower r i g h t  corner i s a v a r i a t i o n o f t h a t seen i n f i g . 15 i n s e t . Uranyl acetate and l e a d c i t r a t e s t a i n i n g .  X 73,200.  48  Figure 1 9 i  Germinal e p i t h e l i a l c e l l and  multivesicular  (arrows). i n the  bodies c o n t a i n i n g  ferritin  Free f e r r i t i n p a r t i c l e s are  u n d e r l y i n g basement membrane.  acetate s t a i n i n g .  Figure 2 0 i  (above) showing v e s i c l e s  X  vesicles  The  Uranyl  24,300.  Overview of g e r m i n a l e p i t h e l i u m (GE) tissues.  a l s o seen  presence of l a r g e  and  subjacent  ferritin-containing  (arrows) i n f i b r o b l a s t s (F) of the  a l b u g i n e a o v a r i i i s w e l l demonstrated. acetate s t a i n i n g .  X  19,400.  tunica  Uranyl  F i g u r e 21i  F e r r i t i n i s present (E),vesicles  (arrows), and i n l u m i n a l plasma  (asterisk).  Note p l a t e l e t  capillary.  F i g u r e 22J  i n capillary endothelial c e l l  (P) w i t h i n lumen o f  Uranyl acetate s t a i n i n g .  A p i c a l s u r f a c e o f germinal  X 30,500.  epithelial cell.  p o s s i b l e sequence o f the p i n o c y t o t i c events u n d e r l y i n g f e r r i t i n uptake i s shown  (arrows).  U r a n y l a c e t a t e and l e a d c i t r a t e s t a i n i n g . X 88,400.  A  F i g u r e 23*  Overview showing germinal e p i t h e l i u m (GE), tunica albuginea o v a r i i , surrounding f o l l i c u l a r  and g r a n u l o s a  cavity (FC).  a c e t a t e and l e a d c i t r a t e s t a i n i n g . I n s e t shows f e r r i t i n i n f o l l i c u l a r ^  high magnification.  cells  Uranyl X 4,700.  f l u i d at  Uranyl a c e t a t e and l e a d  c i t r a t e s t a i n i n g . X 117,000.  55  DISCUSSION The fine structure of rat ovarian germinal epithelium as described in this study compares quite well with that of species previously reported (Wischnitzer, 19^5 ~ mouse;  Gondos, 1969 -  rabbit;  Weakley, 1969 - hamster;  Papadaki and Beilby, 1971 -  human).  No major ultrastructural differences were encountered  in comparing the germinal epitheliumo.ef pre- and postpubertal animals.  This statement applies both to control and to tracer-  exposed animals. HRP and ferritin travel across the epithelium by two predominantly different routes.  HRP movement is mainly extra-  cellular in both pre- and postpubertal animals, whereas ferritin transport is intracellular in both groups. The following discussion examines several major features of normal germinal epithelial ultrastructure, subsequent to which are described the methods of transfer of HRP and ferritin. A.  Germinal Epithelial Cell Shape Variation in germinal epithelial cell shape from squamous  to cuboidal may be explained on the basis of follicular growth. Presumably, the squamous cells represent "stretched" cuboidal cells.  The cuboidal cells may deform according to the stresses  imposed upon them by cell growth and multiplication occurring in underlying developing follicles.  Such Follicular, and hence  ovarian, volume increase would imply a concurrent increase in ovarian surface area.  This is especially marked in the rat,  as it is polyovular, with a number of follicles developing to  56  maturity  simultaneously.  ovary i s t o remain covered  The i n c r e a s e i n s u r f a c e area, i f the by the germinal  a change i n e p i t h e l i a l c e l l shape.  epithelium, requires  The nature  i s an i n c r e a s e i n the area covered by a c e l l , "stretch"' from a c u b o i d a l t o a squamous shape. t h a t the c o n v o l u t e d  o f t h i s change i . e . , a c e l l must I t i s possible  i n f o l d i n g s o f the i n t e r c e l l u l a r  clefts  between c u b o i d a l c e l l s may c o n t r i b u t e t o the r e q u i r e d i n c r e a s e i n c e l l s u r f a c e area.  This i s d i f f i c u l t  c l e f t s between squamous c e l l s are s t i l l  t o a s s e s s , as the q u i t e convoluted.  They  do not appear t o have become more d i r e c t i n t h e i r course, as might be expected. Some authors  suggest t h a t perhaps an i n c r e a s e i n e p i t h e l i a l  c e l l number i s a l s o i n v o l v e d i n e p i t h e l i a l accommodation to o v a r i a n expansion ( W i s c h n i t z e r , 1965? Weakley, 1969). no m i t o t i c stages germinal  other than interphase were observed i n the  e p i t h e l i u m o f t h i s study,  follicles,  However,  even a t the apex o f G r a a f i a n  where such d i v i s i o n might be most expected.  I t thus  seems u n l i k e l y t h a t e p i t h e l i a l c e l l m u l t i p l i c a t i o n c o u l d be a major response t o an i n c r e a s e i n o v a r i a n s u r f a c e area. not t o imply  t h a t the e p i t h e l i a l c e l l s have l o s t t h e i r  This i s capability  to r e p l i c a t e by m i t o t i c d i v i s i o n , merely t h a t t h i s would n o t seem t o be a normal response t o o v a r i a n volume i n c r e a s e . e p i t h e l i a l c e l l s are s t i l l  That  able t o undergo m i t o s i s i s i n d i c a t e d  by the presence o f c e n t r i o l e s i n a s s o c i a t i o n with the c i l i a seen i n the p r e s e n t study.  Subsequent t o o v u l a t i o n the e p i t h e l i a l  c e l l s must presumably undergo m i t o s i s i n order t o r e - e s t a b l i s h the i n t e g r i t y o f the epithelium over the corpus luteum.  Otherwise,  57  regions  denuded of e p i t h e l i a l c e l l s s h o u l d  common occurrence.  have been of q u i t e  To the c o n t r a r y , no such r e g i o n s  were  observed i n the specimens examined. As w e l l as a p a s s i v e  alteration in epithelial c e l l  shape,  r e c e n t s t u d i e s have i m p l i c a t e d the p o s s i b l e involvement of a c t i v e component (Jeppesen, 1975)• by the o b s e r v a t i o n  i n the p r e s e n t  some g e r m i n a l e p i t h e l i a l c e l l s , may  have an  intrinsic  re-establishment  ability  Such a hypothesis  i s supported  study of m i c r o f i l a m e n t s  which suggests t h a t the  to contract.  of cuboidal c e l l  in cells  Post-ovulatory  shape c o u l d thus be  on the b a s i s of a c o n t r a c t i l e event.  an  explained  As a c o r o l l a r y to  this,  c e l l " s t r e t c h i n g " c o u l d be a f u n c t i o n of the r e l a x a t i o n o f an i n t e r n a l system of c o n t r a c t i l e elements.  Microfilaments  are abundant i n the germinal e p i t h e l i a l c e l l s of the guinea p i g ovary, where i t has  been proposed t h a t they c o n t r a c t  the s u p e r f i c i a l l a y e r o f the s t r a t i f i e d e p i t h e l i u m 1975).  The  fetal  (Jeppesen,  r e s u l t o f t h i s c o n t r a c t i o n i s t h a t the more b a s a l l y  s i t u a t e d c e l l s are f o r c e d i n t o the gonadal anlage where they become a s s o c i a t e d with the  sex cords as p r e f o l l i c u l a r  Jeppesen noted t h a t m i c r o f i l a m e n t s i n c e l l shape are e x p l a i n why  epithelial cells, its  development.  B.  Intercellular  are observed only when changes  o c c u r r i n g d u r i n g development.  microfilaments  cells.  This  could  are not o f t e n seen i n a d u l t g e r m i n a l  as the e p i t h e l i u m has  e s s e n t i a l l y completed  Junctions  J u n c t i o n a l s p e c i a l i z a t i o n s of types s i m i l a r to those  58  observed i n r a t germinal e p i t h e l i u m have been noted i n p r e v i o u s s t u d i e s o f both g e r m i n a l e p i t h e l i u m and mesothelium. regions  Focal  o f apparent membrane f u s i o n observed i n t h i s study  Results)  (see  appear s i m i l a r to j u n c t i o n s which have been i d e n t i f i e d  i n the g e r m i n a l e p i t h e l i u m of o t h e r s p e c i e s as t i g h t  junctions .  or j u n c t i o n a l complexes (Papadaki and B e i l b y , 1 9 7 1 ) .  "typical  j u n c t i o n a l complexes ( t e r m i n a l b a r s ) " (Gondos, 1 9 6 9 ) . or t e r m i n a l bars ( W i s c h n i t z e r , describe  1965).  These terms are u s u a l l y r e s e r v e d  j u n c t i o n s which are, or have as one  c i r c u m c e l l u l a r zones of molecular  to  o f t h e i r components, Weakley ( 1 9 6 9 )  occlusion.  was  e x p l i c i t i n a v o i d i n g the term " t i g h t j u n c t i o n " when d e s c r i b i n g the  j u n c t i o n s of hamster germinal e p i t h e l i u m .  f o c a l j u n c t i o n s , she did  As w e l l as  fused  a l s o noted f o c a l r e g i o n s where membranes  not seem to be f u s e d but were separated  by a s m a l l gap  of  o  about 20 A,  s i m i l a r to the s i t u a t i o n observed i n many o f the  f o c a l j u n c t i o n s of the present assess  study.  It is d i f f i c u l t  to  ?  these j u n c t i o n s i n s t r u c t u r a l , and hence f u n c t i o n a l ,  terms with r e g a r d to the degree of membrane s e p a r a t i o n without r e s o r t i n g to more s p e c i a l i z e d techniques.  For  instance,  f r e e z e f r a c t u r e s t u d i e s of the f o c a l j u n c t i o n s which seem t o be separated  by a s m a l l i n t e r c e l l u l a r space c o u l d i n d i c a t e whether  or not these j u n c t i o n s are gap  junctions.  The  appearance o f  membrane f u s i o n c o u l d i n some cases be a r t e f a c t u a l , due  to  o b l i q u i t y of s e c t i o n i n g through a j u n c t i o n , compounded by t o r t t p s i t y of the i n t e r c e l l u l a r c l e f t s . c o u l d indeed  the  Such an i n t e r p r e t a t i o n  e x p l a i n some of the f o c a l f u s i o n of  adjacent  germinal e p i t h e l i a l c e l l membranes seen i n t h i s study.  However,  59  examination of s e r i a l s e c t i o n s a l s o suggests t h a t some of these f o c a l p o i n t s of f u s i o n are i n f a c t q u i t e r e a l and not a r t e f a c t u a l . Such f u s i o n i s not e x t e n s i v e and i t i s d o u b t f u l t h a t i t i s continuous about the e n t i r e p e r i p h e r y of a c e l l as an o c c l u d i n g junction.  As w e l l , i f some germinal e p i t h e l i a l c e l l s are  by z o n u l a r t i g h t  joined  j u n c t i o n s , then one would expect t o observe  such j u n c t i o n s i n a l l i n t e r c e l l u l a r c l e f t s i f the e p i t h e l i u m i s to c o n s t i t u t e an e f f e c t i v e b a r r i e r t o d i f f u s i o n . i n t e r c e l l u l a r c l e f t s devoid of any are  In f a c t ,  junctional specialization  sometimes noted. In  l i g h t of the r e s u l t s of the present study with r e g a r d t o  t r a c e r movement, i t i s extremely u n l i k e l y t h a t the areas o f e p i t h e l i a l c e l l c o n t a c t are t i g h t j u n c t i o n s i n the sense they prevent i n t e r c e l l u l a r m o l e c u l a r d i f f u s i o n .  The  that  interpreta-  t i o n g i v e n t o the f o c a l f u s i o n of membranes seen i n t h i s study i s thus the same as t h a t g i v e n t o j u n c t i o n s o f s i m i l a r i n mesothelium  (Cotran and Karnovsky,  t h e l i u m (Karnovsky,  196?).  1968)  appearance  and c a p i l l a r y endo-  Namely, these s m a l l p o i n t s of  membrane f u s i o n are most l i k e l y maculae o c c l u d e n t e s . or "spots of  occlusion."  T h e i r f u n c t i o n would thus seem t o p r i m a r i l y  i n v o l v e c e l l adherence  r a t h e r than r e g u l a t i o n of m o l e c u l a r  movement through the i n t e r c e l l u l a r spaces o f the germinal e p i thelium. T e l e o l o g i c a l l y , one might reason t h a t the germinal e p i t h e l i u m does not c o n s t i t u t e an impervious b a r r i e r which p r o t e c t s the ovary and i t s c o n t a i n e d oocytes because  the same j u n c t i o n s on  which the e f f e c t i v e n e s s o f such a b a r r i e r depends c o u l d a l s o  60 make o v u l a t i o n a more d i f f i c u l t  process.  That i s , such  junctions  c o u l d c o n s t i t u t e a mechanical hindrance t o the r e l e a s e of oocytes. Even i f such a b a r r i e r d i d a l l o w o v u l a t i o n , every time an oocyte was  r e l e a s e d p o t e n t i a l l y harmful substances c o u l d e n t e r the  ovary.  These substances c o u l d d i f f u s e from the p e r i t o n e a l  c a v i t y through the e p i t h e l i a l break a t the apex of the r u p t u r e d Graafian f o l l i c l e  from which the oocyte was  expelled.  substances would then be i n the c a v i t y o f the corpus  The luteum,  and u n l e s s a l l the c o n s t i t u e n t c e l l s o f t h a t s t r u c t u r e were j o i n e d by o c c l u d i n g j u n c t i o n s , which they do not seem t o be, m o l e c u l a r movement through the ovary c o u l d c o n t i n u e unhindered. T h i s would d e f e a t the whole purpose  of an impermeable e p i t h e l i u m ,  The s i t u a t i o n would be exacerbated i n the r a t , which i s p o l y o v u l a r , and would thus p r e s e n t numerous openings i n t o the ovary from the p e r i t o n e a l c a v i t y through s e v e r a l r u p t u r e d G r a a f i a n follicles. C.  Microvilli The presence of m i c r o v i l l i has been noted i n p r e v i o u s  s t u d i e s of the germinal e p i t h e l i u m ( W i s c h n i t z e r , 1965.' Gondos, 1969;  Weakley, 1969? Papadaki and B e i l b y , 1971), as w e l l as i n  the p r e s e n t study. mesothelium,  Studies of m i c r o v i l l i of the p e r i t o n e a l  which i s o f the same e m b r y o l o g i c a l o r i g i n as the  germinal e p i t h e l i u m , have a l s o been conducted Karnovsky,  1968j Andrews and P o r t e r , 1973).  (Cotran and Most authors b e l i e v e  t h a t m i c r o v i l l i serve to enhance the a b s o r p t i v e or s e c r e t o r y surface-to-volume r a t i o o f a c e l l .  I f t h i s i s the case, i t  61  would indicate that the germinal epithelium, by v i r t u e of i t s numerous m i c r o v i l l i , may be involved i n the translocation of material e i t h e r to or from the peritoneal cavity. Another i n t e r e s t i n g proposal f o r the function of peritoneal m i c r o v i l l i has been put forth by Andrews and Porter (1973)' They believe that the regions between adjacent m i c r o v i l l i entrap the slippery mucins secreted by the peritoneal mesothelial cells.  The exudate may be held either by purely  physical means or by binding of the aqueous component of the exudate by negatively charged acid mucopolysaccharides which coat the c e l l free surface.  "The r e s u l t of such entanglement  and binding would be a slippery l i q u i d cushion layer which could function i n protecting the underlying thin mesothelium from surface abrasion a r i s i n g from normal movement of internal organs over one another."  Such a mechanism could a s s i s t i n the  maintenance of the i n t e g r i t y of the germinal epithelium. Even though the epithelium may be protected i n part from gross damage by the periovarian bursa, movement of the ovary within the bursa would s t i l l necessitate the presence of m i c r o v i l l i . This theory also allows the postulation of one possible function f o r the proteins presumably synthesized and secreted by the germinal e p i t h e l i a l c e l l s .  These proteins could be a component  of the serous exudate which lubricates the surfaces of the intraperitoneal v i s c e r a . Although no s p e c i f i c attempts were made to preserve or s t a i n such a mucinous surface coating i n the present study, there d i d from time to time appear to be amorphous remnants  62  o f such a c o a t i n g adherent to the p e r i t o n e a l s u r f a c e of germinal D.  epithelium.  Protein The  Synthesis  g e r m i n a l e p i t h e l i a l c e l l s are r i c h  r e t i c u l u m , mitochondria, are a l s o present. s y n t h e t i c metabolic with  the  i n rough endoplasmic  and f r e e ribosomes.  G o l g i complexes  These f e a t u r e s are o f t e n a s s o c i a t e d activity,  l i p i d metabolism.  some of which c o u l d be  with  concerned  In a d d i t i o n , the abundance of ribosomes  and rough endoplasmic r e t i c u l u m i n d i c a t e s a high degree of peptide  synthesis.  U s u a l l y , when ribosomes occur s i n g l y  within  the cytoplasm r a t h e r than as polysomes or rough endoplasmic reticulum, use  the manufactured p e p t i d e s  (Lentz, 1971).  are r e t a i n e d f o r endogenous  Rough endoplasmic r e t i c u l u m i n a c e l l i s  commonly concerned with the manufacture of p r o t e i n f o r export (Lentz, 1971).  These observations  those a s s o c i a t e d with suggestive  the endoplasmic r e t i c u l u m are  and  together  of a h i g h i n t r i n s i c metabolism^of the germinal  e p i t h e l i a l c e l l s coupled export.  on both f r e e ribosomes  The  with the s y n t h e s i s of p r o t e i n f o r  abundance of mitochondria  a l s o a t t e s t s to the  high  energy requirements o f these c e l l s , presumably f o r s y n t h e t i c or t r a n s p o r t f u n c t i o n s . export and  In c e l l s whose prime f u n c t i o n i s p r o t e i n  the G o l g i complex i s prominent as the s i t e of packaging  c o n c e n t r a t i o n of p r o t e i n s s y n t h e s i z e d  plasmic  reticulum.  i n the rough endo-  P r o t e i n s are subsequently t r a n s p o r t e d  r e l e a s e d i n the form of membrane bound v e s i c l e s .  As  and  previously  noted (see R e s u l t s ) , the morphology of the G o l g i complex v a r i e s  63  c o n s i d e r a b l y from one germinal e p i t h e l i a l c e l l to  another.  Numerous s m a l l v e s i c l e s of v a r i a b l e e l e c t r o n d e n s i t y and content are o f t e n seen i n the v i c i n i t y of the G o l g i complex.  Because  such v e s i c l e s are a l s o seen to be i r r e g u l a r l y s c a t t e r e d t h r o u g h out much of the r e s t o f the cytoplasm,  it  i s d i f f i c u l t to assess  whether or not they are a l s o a product o f the a c t i v i t y of the G o l g i complex. Weakley (1969) observed the i n d e n t a t i o n o f the b a s a l  surface  o f the g e r m i n a l e p i t h e l i a l c e l l s of the hamster by f o l l i c u l a r c e l l processes  c o n t a i n i n g many ribosomes.  There seemed to be  no i n t e r v e n i n g basement membrane and the c e l l s were a p p a r e n t l y i n direct contact.  She suggested t h a t t h i s c o u l d i n d i c a t e  e i t h e r amino a c i d t r a n s f e r from e p i t h e l i a l to f o l l i c u l a r or p r o t e i n t r a n s f e r plasmic processes  i n the o p p o s i t e d i r e c t i o n .  cells,  No such c y t o -  were observed i n the p r e s e n t s t u d y .  Considering  the amount o f peptide s y n t h e t i c machinery i n the germinal e p i thelial cells,  i t seems u n l i k e l y t h a t these c e l l s would  f r e e amino a c i d s r a t h e r  than completed p e p t i d e s .  transfer  A l s o i n view  o f t h i s e p i t h e l i a l preponderance of s y n t h e t i c o r g a n e l l e s and t h e i r presumed p r o d u c t i o n o f a l a r g e number of p e p t i d e s ,  it  seems u n l i k e l y t h a t f o l l i c u l a r c e l l s would be t r a n s p o r t i n g pept i d e s to the g e r m i n a l e p i t h e l i a l c e l l s . much more reasonable  I t would seem to be  to suspect p r o t e i n t r a n s f e r  the g e r m i n a l e p i t h e l i a l c e l l s  to e i t h e r the u n d e r l y i n g f o l l i c u l a r  c e l l s or to the o v e r y l i n g p e r i t o n e a l c a v i t y . i n t r a c e l l u l a r o r g a n e l l e s i s not c o n s i s t e n t any major route o f s e c r e t o r y  to occur from  activity.  The p o l a r i t y of  enough  to  indicate  64  Autoradiographic  s t u d i e s may prove u s e f u l i n f u r t h e r  i n v e s t i g a t i o n o f p r o t e i n s y n t h e s i s , t r a n s p o r t , and f u n c t i o n . E.  Lipids L i p i d i n c l u s i o n s s i m i l a r t o those seen i n t h i s study have  a l s o been observed i n the g e r m i n a l e p i t h e l i a l c e l l s o f the hamster (Weakley, 1 9 6 9 )  1971).  and human (Papadaki and B e i l b y ,  I t i s p o s s i b l e t h a t such i n c l u s i o n s a r e a s s o c i a t e d with s t e r o i d a l activity  s i n c e t h e i r morphology i s s i m i l a r t o t h a t observed i n  subjacent  cortical cells.  Germinal e p i t h e l i a l c e l l s a r e b e l i e v e d  to give r i s e t o f o l l i c u l a r c e l l s d u r i n g degree o f s t e r o i d a l a c t i v i t y cells.  development.  i s manifest i n these  although  the r e t e n t i o n o f an e p i t h e l i a l  c a p a b i l i t y f o r s t e r o i d metabolism (Weakley, 1 9 6 9 ? Beilby,  follicular  S i m i l a r a c t i v i t y i n the germinal e p i t h e l i u m ,  not as pronounced, c o u l d r e p r e s e n t  A high  Papadaki and  1971).  C e r t a i n enzymes have been h i s t o c h e m i c a l l y l o c a l i z e d germinal e p i t h e l i a l c e l l s .  The c o n c e n t r a t i o n  s t e r o i d dehydrogenase i s q u i t e marked.  o f 173-hydroxy-  A l s o present  16-a and 166 forms, the l e v e l s of which are much lower et a l . , 1 9 6 6 ) .  within  a r e the (Baillie  These enzymes are c a t a l y t i c f o r s p e c i f i c  i n the metabolism o f 1 6 - and 1 7 - h y d r o x y s t e r o i d s , probably o f an e s t r o g e n i c  reactions  which a r e  nature ( B a i l l i e e t a l . , 1 9 6 6 ) .  It  i s d i f f i c u l t t o r e c o n c i l e the presence of these enzymes w i t h i n the g e r m i n a l e p i t h e l i a l c e l l s , as the c e l l s have n o t been found to demonstrate  3-hydroxysteroid  (Baillie et a l . , 1966).  dehdrogenase a c t i v i t y  T h i s i s an e s s e n t i a l enzyme i n the  65  i n i t i a l stages  of metabolism of the'16- and  17-hydroxysteroids.  I t would seem to be h i g h l y u n l i k e l y t h a t the enzymes o f l a t e r stages  of the metabolic  pathways would be present  i n the  absence of the enzyme r e q u i r e d to s y n t h e s i z e t h e i r p r e c u r s o r s . I t may  be t h a t the h i s t o c h e m i c a l techniques  investigate  8-hydroxysteroid  used to  dehydrogenase a c t i v i t y  not s e n s i t i v e enough to r e v e a l i t s presence i n the epithelial cells. metabolic finally,  The  are  germinal  p o s s i b i l i t y a l s o e x i s t s of an a l t e r n a t e  pathway which does not r e q u i r e t h i s enzyme. the s u b s t r a t e s f o r 173-»  16a-,  and  l6g-hydroxysteroid  dehydrogenase c a t a l y s i s c o u l d be s y n t h e s i z e d i n other and t r a n s f e r r e d to the germinal  And  cells  epithelial cells for final  processing. Weakley (1969) noted t h a t i n the germinal  e p i t h e l i u m of the  hamster there i s an i n c r e a s e i n the number of l i p i d i n c l u s i o n s with development.  Such i n c l u s i o n s are present  t i s s u e s but u s u a l l y disappear  i n many embryonic  during d i f f e r e n t i a t i o n .  Because  o f the i n c r e a s e i n l i p i d i n c l u s i o n s i n the e p i t h e l i a l  cells,  she suggested t h a t "they r e p r e s e n t a s p e c i f i c product  of  d i f f e r e n t i a t e d c e l l r a t h e r than n u t r i e n t m a t e r i a l to be by the developing  tissue."  the used  The presence o f l i p i d i n c l u s i o n s ,  t h e i r i n c r e a s i n g numbers d u r i n g development, and the t i o n o f s p e c i f i c enzymes w i t h i n the germinal  localiza-  epithelial  cells  a l l p o i n t to a d e f i n i t e e p i t h e l i a l involvement i n s t e r o i d metabolism.  However, t h i s involvement i s presumably of a  much l e s s e r degree than i s seen i n f o l l i c u l a r and cells.  luteal  T h i s i s a t t e s t e d to by the lower cytoplasmic  lipid  66  content  of e p i t h e l i a l c e l l s and by e i t h e r the absence or very  s m a l l number of u l t r a s t r u c t u r a l f e a t u r e s u s u a l l y a s s o c i a t e d with c e l l s whose prime f u n c t i o n i s s t e r o i d o g e n i c .  These l a t t e r  f e a t u r e s i n c l u d e t u b u l a r m i t o c h o n d r i a l c r i s t a e and  smooth  endoplasmic r e t i c u l u m . plasmic  r e t i c u l u m may  I t i s p o s s i b l e t h a t some smooth endohave been mistaken f o r s m a l l i n t r a c e l l u l a r  vesicles. F.  HRP  Movement and  Localization  The  r e s u l t s of the present study i n d i c a t e  t h a t the  e p i t h e l i u m o f the r a t i s r e a d i l y permeable to HRP. ment o f HRP clefts.  germinal  The move-  seems to be predominantly through the i n t e r c e l l u l a r  V e s i c u l a r t r a n s p o r t o f HRP  a c r o s s the germinal  epi-  thelium c o u l d not be proven, even though there does seem to be a s m a l l but d e f i n i t e p o p u l a t i o n of t r a c e r - f i l l e d cytoplasmic v e s i c l e s which do not appear to be connected to any surfaces.  However, i n terms of the numbers of these  t h e i r s i z e , and t h e i r HRP vesicular  content,  t r a n s p o r t i n moving HRP  s i z e across  the germinal  intercellular diffusion.  vesicles,  e p i t h e l i u m i s minor i n comparison to These r e s u l t s are s i m i l a r to  those  of r a t  o r i g i n to the  epithelium.  The movement of HRP i n the nature  of  and other molecules of a s i m i l a r  mesothelium, which i s o f s i m i l a r e m b r y o l o g i c a l  probably  cell  i t would seem t h a t the r o l e  found by Cotran and Karnovsky (1968) i n t h e i r study  germinal  o f the  through the i n t e r c e l l u l a r c l e f t s i s of a p a s s i v e d i f f u s i o n process.  i s l e n t to t h i s c o n c l u s i o n by the work of Cotran and  Support  Karnovsky  67  (1968) on mesothelium, which i s u l t r a s t r u e t u r a l l y , and thus presumably p h y s i o l o g i c a l l y , q u i t e s i m i l a r t o the germinal epithelium.  In mesothelium exposed t o HRP e i t h e r d u r i n g or  a f t e r f i x a t i o n there was s t i l l HRP.  i n t e r c e l l u l a r l o c a l i z a t i o n of  Any a c t i v e HRP-transporting mechanism i n the i n t e r c e l l u l a r  c l e f t s would o b v i o u s l y have been rendered thereby l e a v i n g simple  non-functional,  d i f f u s i o n as the only means by which  HRP c o u l d be t r a n s l o c a t e d .  Small v e s i c l e s f i l l e d with  HRP  i n t h i s same study were always d i r e c t l y connected t o the a p i c a l s u r f a c e o r i n t e r c e l l u l a r c l e f t s or were i n c l o s e a p p o s i t i o n t o them, again s u g g e s t i v e  of f i l l i n g by d i f f u s i o n (see R e s u l t s ) .  There were no t r a c e r - c o n t a i n i n g v e s i c l e s deep w i t h i n the mesothelial c e l l s . bility  In a d d i t i o n , s t u d i e s o f f o l l i c u l a r permea-  t o i n t r a v a s c u l a r l y - i n j e c t e d HRP have shown t h a t HRP may  appear i n the i n t e r c e l l u l a r c l e f t s o f f a t germinal e p i t h e l i u m a f t e r l e a v i n g the c i r c u l a t i o n  (Anderson, W.,  1972a).  Thus  there i s movement o f HRP not o n l y from the basement membrane toward the p e r i t o n e a l s u r f a c e b u t a l s o i n the opposite t i o n , as r e v e a l e d i n the present  study.  direc-  To e x p l a i n these move-  ments on the b a s i s o f the o p e r a t i o n of a c t i v e t r a n s p o r t mechanisms, one. would have t o p o s t u l a t e the e x i s t e n c e  o f e i t h e r an  a c t i v e t r a n s p o r t mechanism moving the same type o f molecule bidirectionally.  A l t e r n a t i v e l y , two d i f f e r e n t mechanisms, each  t r a n s p o r t i n g the same type o f molecule, b u t i n d i f f e r e n t d i r e c t i o n s , c o u l d be p o s t u l a t e d .  In e i t h e r case t h i s i s n o t  likely,  as i t would be o f no obvious b e n e f i t t o a c e l l o r  tissue.  The r e s u l t s do suggest t h a t HRP movement i n the  68  i n t e r c e l l u l a r c l e f t s i s not r e s t r i c t e d as to d i r e c t i o n , and would thus seem t o support a d i f f u s i o n theory f o r HRP movement through the germinal e p i t h e l i u m . The p o s s i b i l i t y that the i n t e r c e l l u l a r c l e f t s may have been f i l l e d with t r a c e r by v e s i c l e s from the a p i c a l s u r f a c e  emptying  t h e i r contents i n t o the c l e f t s has a l r e a d y been noted,(see Results).  T h i s f a c t o r was a l s o taken i n t o account by C o t r a n  and Karnovsky  (1968).  They showed t h a t exposure o f mesothelium  to HRP e i t h e r d u r i n g or subsequent  to i n i t i a l  f i x a t i o n (and  p r e c e d i n g p o s t - f i x a t i o n ) . w h i c h would h a l t v e s i c u l a r t r a n s p o r t , did  n o t measurably  clefts.  reduce the amount o f HRP i n the i n t e r c e l l u l a r  These r e s u l t s demonstrate  t h a t i f there i s a v e s i c u l a r  contribution to c l e f t f i l l i n g i t i s inconsequential. the  same may h o l d t r u e f o r the germinal e p i t h e l i u m .  G.  F e r r i t i n Movement and L o c a l i z a t i o n The r e s u l t s suggest t h a t the movement o f f e r r i t i n  the  Presumably  through  germinal e p i t h e l i u m i s an i n t r a c e l l u l a r process mediated  by v e s i c u l a r t r a n s p o r t .  A s i m i l a r cytoplasmic v e s i c u l a r  trans-  p o r t o f f e r r i t i n i s the s o l e means of f e r r i t i n movement through c a p i l l a r y and e n d o t h e l i a l c e l l s subsequent f e r r i t i n injections  (Anderson, W.,  i s no evidence whatsoever  to intravascular  1972a; Payer, 1975)-  o f i n t e r c e l l u l a r passage  There  of f e r r i t i n  through the germinal e p i t h e l i u m . I t has been noted t h a t f r e e f e r r i t i n p a r t i c l e s are occas i o n a l l y seen w i t h i n e p i t h e l i a l c e l l s and f i b r o b l a s t s and apparently i n i n t e r c e l l u l a r c l e f t s .  T h e i r appearance i n  69  these l o c a t i o n s i s regarded as a r t e f a c t u a l f o r a number of reasons.  Firstly,  i n t h i s study.  these s i t u a t i o n s were observed  infrequently  I f the cytoplasm o r the i n t e r c e l l u l a r  clefts  were common s i t e s o f f e r r i t i n movement, one would expect t o observe f r e e i n t r a c y t o p l a s m i c p a r t i c l e s on a c o n s t a n t b a s i s .  and i n t e r c e l l u l a r  ferritin  Such i s not the case.  a d d i t i o n , f r e e f e r r i t i n p a r t i c l e s , when p r e s e n t  In  within a c e l l ,  are n o t l i m i t e d i n occurrence t o the c y t o p l a s m i c matrix.  They  may appear i n d i s c r i m i n a t e l y both w i t h i n the m a t r i x and w i t h i n most c e l l o r g a n e l l e s organelles.  and i n o r on the membranes o f those  This suggests t h a t f e r r i t i n p a r t i c l e s may have  been "smeared" across The  a s e c t i o n and are s u p e r f i c i a l l y s i t u a t e d .  source of these p a r t i c l e s cannot be d e f i n i t e l y  They c o u l d be d e r i v e d from s e c t i o n e d l a r g e numbers o f f e r r i t i n molecules. may have been d i s p l a c e d d u r i n g  established.  v e s i c l e s which  contain  Some o f these molecules  the s e c t i o n i n g process t o sub-  s e q u e n t l y appear randomly over a s e c t i o n .  Excessive  f e r r i t i n at  the p e r i t o n e a l s u r f a c e may a l s o have been d i s p l a c e d by s e c t i o n i n g . F i n a l l y , the d e n s i t y s t a n t i a l l y greater  of i n t r a v e s i c u l a r f e r r i t i n  than the d e n s i t y  of f r e e f e r r i t i n ,  the overwhelming predominance o f v e s i c u l a r The  penetration  j a c e n t regions  i s always subindicating  transport.  of f e r r i t i n through the e p i t h e l i u m  i s much slower than the movement of HRP.  and subThis  i s l i k e l y a f u n c t i o n o f t h e i r d i f f e r i n g m o l e c u l a r s i z e and weight and consequent d i f f e r e n c e s i n t h e i r modes o f movement. Such temporal d i f f e r e n c e s have been noted i n other employing these two t r a c e r s  (Payer, 1975).  studies  70  The presence of f e r r i t i n i n the f o l l i c u l a r f l u i d o f some developing f o l l i c l e s  i n d i c a t e s t h a t the f o l l i c u l a r basement  membrane does n o t c o n s t i t u t e a b a r r i e r t o f e r r i t i n movement. T h i s c o n t r a d i c t s the r e s u l t s of a p r e v i o u s study (Anderson,  W.,  1972a).  Using the same s t r a i n o f r a t as was used i n t h i s study,  Anderson  found t h a t i n t r a v a s c u l a r l y - i n j e c t e d f e r r i t i n seemed  t o accumulate  a t the l e v e l o f the f o l l i c u l a r basement membrane  and d i d not pass through i t .  No e x p l a n a t i o n can be put f o r t h  a t p r e s e n t t o r a t i o n a l i z e the observed d i f f e r e n c e s i n f o l l i c u l a r permeability.  The d i f f e r e n c e s do not seem t o be r e l a t e d to the  time o f exposure  to f e r r i t i n ,  as the times i n the p r e s e n t study  and i n Anderson's study were s i m i l a r . Anderson  (W., 1972a), i n h i s study o f f o l l i c u l a r p e r m e a b i l i t y ,  used a v a r i e t y o f i n t r a v a s c u l a r t r a c e r s .  He shows the presence  o f T h o r o t r a s t (molecular diameter 70 A) i n i n t r a c e l l u l a r  clefts  and l a r g e p h a g o c y t i c vacuoles of the germinal e p i t h e l i u m . These vacuoles a r e i n c l o s e p r o x i m i t y t o the c l e f t s and some appear as though i n these c l e f t s  they c o u l d be s e c t i o n s through (see R e s u l t s ) .  irregularities  I t i s suggested by Anderson  that  T h o r o t r a s t does n o t pass through the e n t i r e l e n g t h o f the clefts  i n t o the p e r i t o n e a l c a v i t y , due t o the presence o f  j u n c t i o n s i n the c l e f t s .  N e v e r t h e l e s s , f o c a l and extended  j u n c t i o n s were o f t e n observed a t the b a s a l end of i n t e r c e l l u l a r clefts  i n m a t e r i a l examined i n the present study.  Presumably  these types o f j u n c t i o n s would a l s o have been p r e s e n t i n the experimental animals used by Anderson, were of the same s t r a i n as used h e r e i n .  as some o f h i s animals I f T h o r o t r a s t passed  71  e i t h e r through or around these b a s a l l y - s i t u a t e d j u n c t i o n s , i t would be  l i k e l y t h a t i t would a l s o pass through  junctions  a t the p e r i t o n e a l ends of the c l e f t s , as there are no regions  o f o c c l u s i o n there.  A d d i t i o n a l l y , as  mentioned, some c l e f t s appear t o have no zations at a l l . entered  zonal  previously  junctional s p e c i a l i -  ;  I t would thus be expected t h a t i f T h o r o t r a s t  the b a s a l end  of such a c l e f t ,  d i f f u s e to the p e r i t o n e a l end The  then  and  i t would  eventually  i n t o the p e r i t o n e a l c a v i t y .  upper l i m i t o f molecular s i z e i n terms of p a s s i v e  intero  c e l l u l a r d i f f u s i o n would therefore seem to be between 70 (Thorotrast) The  and  110  A  (ferritin).  o v e r a l l d i s t r i b u t i o n of f e r r i t i n i n the c u r r e n t  p a r a l l e l s i n many r e s p e c t s the  t h a t seen i n previous  ovary i n which f e r r i t i n was  i n j e c t i o n (Anderson, W., The  observation  A  introduced  study  studies  of  v i a inatravascular  1972a; Payer, 1975).  t h a t there are more l a r g e v e s i c l e s i n  f i b r o b l a s t s subsequent to f e r r i t i n exposure than are seen i n c o n t r o l m a t e r i a l suggests t h a t f e r r i t i n may e f f e c t on v e s i c u l a r  formation.  have an  inducing  72  CONCLUSION On the b a s i s o f i t s u l t r a s t r u e t u r a l f e a t u r e s , the e p i t h e l i u m appears to be a h i g h l y a c t i v e t i s s u e .  germinal  I t possesses  s t r u c t u r e s s u g g e s t i v e of an a b s o r p t i v e and/or s e c r e t o r y capacity  (microvilli),  a marked p r o t e i n s y n t h e t i c a c t i v i t y  (ribo-  somes and rough endoplasmic r e t i c u l u m ) , and p o s s i b l e s t e r o i d metabolism ( l i p i d i n c l u s i o n s ) .  That the e p i t h e l i u m may  be  a c t i v e l y or p a s s i v e l y i n v o l v e d i n the movement of t r a c e r s has a l s o been demonstrated. Exposure of the p e r i t o n e a l s u r f a c e of the ovary to HRP ferritin,  and  and subsequent e l e c t r o n m i c r o s c o p i c l o c a l i z a t i o n of  these t r a c e r s , i n d i c a t e t h a t there are two  d i s t i n c t routes o f  m o l e c u l a r movement from the p e r i t o n e a l c a v i t y a c r o s s the  germinal  e p i t h e l i u m , a c c o r d i n g to the s i z e of the molecule t r a n s l o c a t e d . o  Molecules  of the order of 40 A diameter,  f r e e l y d i f f u s e through  such as HRP,  appear to  the i n t e r c e l l u l a r c l e f t s of the e p i t h e l i u m . o  Larger molecules,  comparable i n s i z e to f e r r i t i n  (110 A), are  excluded from the c l e f t s and seem to be r e s t r i c t e d to i n t r a c e l l u l a r passage a c r o s s the germinal e p i t h e l i u m .  "Such a cytoplasmic  route c o u l d p r o v i d e a means of s c r e e n i n g substances  destined for  the u n d e r l y i n g t i s s u e s , or of a l t e r i n g them c h e m i c a l l y before they are allowed to proceed only the f e r r i c hydroxide e l e c t r o n microscope,  f u r t h e r (Weakley, 1969)."  core o f f e r r i t i n  i s seen with  i t i s not p o s s i b l e to observe  Because the  whether  changes i n the a p o p r o t e i n p o r t i o n of the f e r r i t i n molecule d u r i n g i t s passage through  the e p i t h e l i u m .  probably be amenable to i n v e s t i g a t i o n by techniques.  occur  Such a study would  immunohistochemical  73  I t can be concluded t h a t there i s ready access o f substances the s i z e o f f e r r i t i n or s m a l l e r i n t o the substance o f the ovary through the germinal e p i t h e l i u m .  From t h i s i t f o l l o w s  t h a t substances which permeate the e p i t h e l i u m and s u b j a c e n t t i s s u e s may,  i f not a l t e r e d d u r i n g t h e i r t r a n s e p i t h e l i a l  i n f l u e n c e the normal development  of the oocyte.  e x e r t e d on the oocyte by these substances may detrimental.  The  or may  passage,  effects not be  At the l e v e l o f the e p i t h e l i u m , s m a l l e r molecules  which move e x t r a c e l l u l a r l y are l i k e l y not s u b j e c t t o any b i o c h e m i c a l s c r e e n i n g procedure which c o u l d e i t h e r prevent t h e i r passage  or a l t e r t h e i r m o l e c u l a r s t r u c t u r e .  e f f e c t s , i f any, on the oocyte would be d i r e c t .  Hence t h e i r Larger molecules,  which must pass through the e p i t h e l i a l c e l l s , c o u l d be s u b j e c t to i n t r a c y t o p l a s m i c m o d i f i c a t i o n of t h e i r s t r u c t u r e , or jtheir passage c o u l d be prevented e n t i r e l y .  The magnitude of the  e f f e c t o f these molecules on the oocyte would be a f u n c t i o n o f the degree o f i n t r a c e l l u l a r a l t e r a t i o n of t h e i r m o l e c u l a r structure. I f an exogenous substance e n t e r s the p e r i t o n e a l c a v i t y , the p o s s i b l e e f f e c t s o f such a substance on the oocyte must be examined a t s e v e r a l s t a g e s .  Firstly,  i n terms o f adverse  e f f e c t s , foreign; molecules c o u l d i n t e r f e r e with normal development  and metabolism.  T h i s c o u l d l e a d to a r e d u c t i o n  i n o o c y t i c v i a b i l i t y and a subsequent t o reach m a t u r i t y .  oocyte  f a i l u r e of the oocyte  Secondly, such molecules c o u l d a l t e r the  oocytes, but i n a l e s s r a d i c a l manner.  Oocytic  development,  o v u l a t i o n , and f e r t i l i z a t i o n c o u l d occur, but the zygote so  ,  74  formed might not be v i a b l e , l e a d i n g t o an i n c r e a s e d r a t e o f spontaneous a b o r t i o n .  F i n a l l y , the e f f e c t s o f exogenous  molecules c o u l d be so s u b t l e as to a l l o w development o f the fertilized  oocyte t o term.  However, the r e s u l t a n t progeny  c o u l d m a n i f e s t c o n g e n i t a l a b n o r m a l i t i e s of v a r y i n g degrees of s e v e r i t y due t o a l t e r a t i o n s i n the oocyte induced by the exogenous molecules. In  terms o f the p o s s i b l e c l i n i c a l s i g n i f i c a n c e o f the  f i n d i n g s o f t h i s study, t h e r e are a number o f s i t u a t i o n s , i n which exogenous molecules may e n t e r the p e r i t o n e a l c a v i t y thus come i n t o c o n t a c t with the germinal e p i t h e l i u m . of  Most  these s i t u a t i o n s are a s s o c i a t e d with the e n t r y of b a c t e r i a  i n t o the p e r i t o n e a l c a v i t y . of  and  The b a c t e r i a are the a c t u a l source  the exogenous molecules, which they r e l e a s e as e i t h e r  exotoxins or endotoxins.  P o s i t i v e i d e n t i f i c a t i o n of some  exotoxins as enzymes has been made and t h e i r composition  and  s i z e found to be s i m i l a r t o enzymes i n g e n e r a l (Davis e t a l . , 1973).  The normal range of enzyme molecular weight  12,000 to more than 1 m i l l i o n (Lehninger, 1970).  i s from  I t would  then appear t h a t of the known b a c t e r i a l e x o t o x i n s , a number are of a molecular weight  l e s s than t h a t of f e r r i t i n  They may thus be able t o pass through  (4§2,000).  the germinal e p i t h e l i u m .  Another source of exogenous molecules c o u l d be the d i g e s t i v e t r a c t subsequent  to p e r f o r a t i o n of an organ.  This would allow  not only b a c t e r i a to e n t e r the p e r i t o n e a l c a v i t y , but a l s o v a r i o u s products of d i g e s t i o n and e x c r e t i o n .  The r o u t e s by  which t o x i n - p r o d u c i n g b a c t e r i a and n o n - b a c t e r i a l l y d e r i v e d  75 exogenous substances may e n t e r the p e r i t o n e a l enumerated below ( E l l i s and Calne, 1.  c a v i t y are  1976)»  from the e x t e r i o r v i a an i n f e c t i o n a t laparotomy or a p e n e t r a t i n g wound.  2.  from i n t r a - a b d o m i n a l a)  viscerat  gangrene of a v i s c u s ,  e.g. acute  appendicitis,  acute c h o l e c y s t i t i s , d i v e r t i c u l i t i s o r i n f a r c t i o n of the i n t e s t i n e , b)  perforation  of a v i s c u s ,  ulcer, perforated  e.g. p e r f o r a t e d  appendicitis,  duodenal  rupture of i n t e s t i n e  from trauma. c) 3.  p o s t - o p e r a t i v e leakage o f an i n t e s t i n a l s u t u r e  v i a the b l o o d stream as p a r t coccal,  4.  streptococcal,  or  line.  of a s e p t i c e m i a (pneumo-  staphylococcal).  v i a the female g e n i t a l t r a c t as i n acute s a l p i n g i t i s or a p u e r p e r a l i n f e c t i o n .  and  I t i s thus seen t h a t  the germinal e p i t h e l i u m i s a  synthetically active  tissue.  metabolically  Whether or n o t i t i s capable o f  m o d i f y i n g some of the molecules t o which i t i s permeable remains to be demonstrated. significance oocytic  Such processes c o u l d be of c o n s i d e r a b l e  i n the maintenance of a normal environment f o r  development, e s p e c i a l l y as there are d e f i n i t e  i n which exogenous molecules may e n t e r the p e r i t o n e a l  situations cavity.  76  LITERATURE CITED Ainsworth, S.K. and M.J. 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