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Ultrastructure of the rat ovarian germinal epithelium and its permeability to electron microscopically.. 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 of ANATOMY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1976 © Ranald Ross Donaldson, 1976 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree l y ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th i s thes is fo r f i nanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Department of ANATOMY The Un ivers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date April 6. 1976 i i ABSTRACT The de t a i l e d fine structure of normal r a t ovarian germinal epithelium was studied by means of transmission and scanning electron microscopy. The germinal epithelium possesses features suggestive of an absorptive and/or secretory capacity, a marked protein synthetic a b i l i t y , and possible s t e r o i d metabolism. These and other c e l l u l a r features were i n turn in d i c a t i v e of a possible germinal 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 of par t i c u l a t e substances. In order to investigate the permeability of the germinal epithelium to molecules from the peritoneal cavity, two electron microscopic tracer molecules, horseradish peroxidase (HRP) and f e r r i t i n , were injected i n t r a p e r i t o n e a l l y . The results indicate that there i s a d i f f e r e n t i a l movement of these two molecules across the germinal epithelium, presumably re l a t e d to the difference; i n t h e i r molecular dimensions. The predominant route of movement of HRP i s e x t r a c e l l u l a r , apparently by d i f f u - sion through the i n t e r c e l l u l a r c l e f t s . Ferritin ( lmovement, on the other hand, i s i n t r a c e l l u l a r , v i a a ves i c u l a r transport mechanism associated with pinocytotic a c t i v i t y at the a p i c a l surface of the germinal e p i t h e l i a l c e l l s . I t i s concluded that the germinal epithelium i s a meta- b o l i c a l l y active tissue which plays both a passive and an active role i n the movement of molecules from the peritoneal cavity. i i i TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i i i LIST OF FIGURES v ACKNOWLEDGEMENT v i INTRODUCTION 1 A. Previous Studies of the Germinal Epithelium .... 1 B. Electron Microscopic Tracers 5 C. Scope of the Present Study . 9 MATERIALS AND METHODS 11 RESULTS 18 A. Normal Germinal Epithelium 18 1. General E p i t h e l i a l Morphology 18 2. C e l l Membrane Features j 19 a. Lateral c e l l membranes* i n t e r c e l l u l a r c l e f t s and junctions • • • • 1 9 b. Apical c e l l membrane 21 c. Basal c e l l membrane 22 3. Nuclear Structure 22 4. Organellar Structure 2 3 B. Tracer Experiments 2 5 1. HRP D i s t r i b u t i o n 2 5 2. F e r r i t i n D i s t r i b u t i o n 30 C. Figures 34 i v Table of Contents (Cont.) Page DISCUSSION 5 5 A. Germinal E p i t h e l i a l C e l l Shape 5 5 B. I n t e r c e l l u l a r Junctions 57 C . M i c r o v i l l i ^ 60 D. Protein Synthesis 62 E. Lipids • 64 F. HRP Movement and Lo c a l i z a t i o n 66 G. F e r r i t i n Movement and Lo c a l i z a t i o n 6 8 CONCLUSION 72 LITERATURE CITED , 76 V LIST OF FIGURES Figures Pages 1-11 Normal germinal epithelium 35-42 12-18 Germinal epithelium exposed to HRP 4 3-48 19-23 Germinal epithelium exposed to 49-54 f e r r i t i n v i ACKNOWLEDGEMENT To Dr. W. A. Webber, my s u p e r v i s o r d u r i n g the c o u r s e o f t h i s s t u d y , I would l i k e 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 . H i s gu i d a n c e , encouragement, and s u p p o r t t h r o u g h o u t a l l s t a g e s o f 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 Drs. W. K. O v a l l e , B. J . P o l a n d , and M. E. Todd f o r t h e i r p a i n s t a k i n g and thorough c r i t i c i s m s and v a l u a b l e s u g g e s t i o n s d u r i n g t h e . p r e p a r a t i o n o f t h i s t h e s i s . F i n a l l y , my thanks go t o Mrs. P a t r i c i a H o l l i n g d a l e , w i t h o u t 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 phases o f t h i s p r o j e c t , i t c o u l d n o t have been completed. T h i s s t u d y was s u p p o r t e d by a S t u d e n t s h i p from the M e d i c a l Research C o u n c i l o f Canada. 1 INTRODUCTION A. Previous Studies of the Germinal Epithelium The functional significance of the germinal epithelium of the mammalian ovary was f i r s t investigated i n terms of i t s possible oogenic p o t e n t i a l (Waldeyer, 1870, quoted by Franchi et a l . , 1962). As c l e a r l y evident by the name accorded i t by early workers, the germinal epithelium was considered to be a source of ovarian germ c e l l s , (Franchi et a l . , 1962). Subse- quent studies have demonstrated that primordial germ c e l l s which give r i s e to d e f i n i t i v e oocytes arise e i t h e r from stem c e l l s which give r i s e to endodermal c e l l s , or from the endoderm of the embryonic secondary yolk sac, near the s i t e of the a l l a n t o i c evagination (Witschi, 19#8). These germ c e l l s then a c t i v e l y migrate i n an amoeboid fashion, perhaps assisted by h i s t i o l y t i c action, to the region of the gonadal blastema by way of the dorsal mesentery of the developing gut (Witschi, 19^8} Pinkerton et a l . , 1961). The gonadal blastema i s i n i - t i a l l y a thickened region of the coelomic mesothelium and underlying mesenchyme on the ventromedial aspect of the urogenital ridges. This mesothelium l a t e r becomes the germinal epithelium. I t i s now well established that a l l d e f i n i t i v e germ c e l l s are progeny of the primordial germ c e l l s which f i r s t populate the ovary. The germinal epithelium does not contribute to the germ c e l l population at any stage of ovarian development (Witschi, 19631 Franchi, 1970). Currently, a number of investigators believe that the 2 germinal epithelium i s active mainly during prenatal, and perhaps early postnatal development. At these times i t i s believed to be a source of precursors of f o l l i c u l a r ( l a t e r , granulosa) c e l l s of the developing ovarian cortex (Franchi, 1970). This i s disputed, however, by others who suggest that the f o l l i c u l a r c e l l s are derived from mesenchymal and not mesothelial (germinal e p i t h e l i a l ) c e l l s (Franchi, 197°)• Apart from the developmental function of the germinal epithelium, i t s f u nctional a c t i v i t y i n the postnatal animal (exclusive of e a r l i e r studies of p o t e n t i a l germinal a c t i v i t y ) has not been serious l y considered. Most recent studies of the germinal epithelium have been concerned with i t s descriptive ultrastrueture, both during intra-uterine and postnatal development (Wischnitzer, 19651 Gondos, 1969i Weakley, 1969» Papadaki and Beilby, 1971; Jeppesen, 1975? Merchant, 19751 P e l l i n i e m i , 1975). Papadaki and Beilby (1971) and Weakley (1969) have suggested that germinal e p i t h e l i a l c e l l s , on the basis of t h e i r u l t r a s t r u e t u r a l features, may transfer material through the cytoplasm. The d i r e c t i o n of movement, however, could not be ascertained from t h i s morphological data alone. These workers observed pino- c y t o t i c v e s i c l e s , vacuoles, m i c r o v i l l i and an abundance of ribosomes, rough endoplasmic reticulum, and mitochondria within the germinal e p i t h e l i a l c e l l s . Since such features are often prominent i n c e l l s able to carry on transport and synthetic processes, they considered t h e i r assumptions warranted. Of those studies of the germinal epithelium conducted to 3 date, only Ghiquoine's work (1961) involved 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 microscopic studies w i l l be mentioned l a t e r ) . To determine whether or not the germinal epithelium had an oogenic function, mice were injected 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 gold, an electron microscopically demonstrable tracer 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 gold and gave r i s e to germ c e l l s (which have d i s t i n c t l y recognizable h i s t o l o g i c a l , h i s t o - chemical, and u l t r a s t r u c t u r a l features), then Chiquoine would have expected to f i n d p a r t i c u l a t e gold tracer within the c e l l s so derived. Because no oocytes were l a b e l l e d , Chiquoine con- cluded that " v i t a l s t a i n i n g of the germinal epithelium provides no evidence f o r an oogenic function on the part of the germinal epithelium i n the postnatal animal." This resolved 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 (Latta and Pederson, 1944j Jones, 19^9), which were l i m i t e d i n part by virtue of inherent technical r e s t r i c t i o n s . Chiquoine's description of tracer 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 that aggregates of gold p a r t i c l e s were randomly scattered within the germinal e p i t h e l i a l c e l l s . No mention was made of how the gold tracer might have entered the c e l l s , nor whether i t s course was followed over a period of time using a series of animals. Zuckerman (195D has also made mention of the uptake of parti c u l a t e matter by the germinal epithelium. He sa i d that i t " i s highly phagocytic, and i n the normal animal picks up debris from red blood c e l l s or any part i c u l a t e matter of sui t a b l y small size that i s introduced into the peritoneal 4 c a v i t y . " Unfortunately, this paper presented no substantiating evidence f o r that statement, nor did i t mention the experimental animal to which that statement referred. Chiquoine's study, although presumably not intended to i l l u s t r a t e tracer movement across the germinal epithelium, did show that germinal e p i t h e l i a l c e l l s can take up c o l l o i d a l gold. This uptake could represent the f i r s t step of a c e l l - mediated transport. However, since then there have been no reports i n the l i t e r a t u r e concerned with tracer movement across the germinal epithelium. Such movement could conceivably be either by passive d i f f u s i o n or by active transport. It could occur across the germinal epithelium i n a 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, either 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 transport properties could be of po t e n t i a l p h y s i o l o g i c a l and developmental significance to the ovary. This applies e s p e c i a l l y to the oocytes i n that c e r t a i n substances, some perhaps of a deleterious nature i n terms of oocyte development, might be able to pass or be transported from the peritoneal c a v i t y into the substance of the ovary. Transport could also be operative over s l i g h t l y shorter distances i n the case of substances a c t u a l l y manufactured within the germinal e p i t h e l i a l c e l l s and transferred to sub- jacent c e l l s of the tunica albuginea o v a r i i and cortex. Weakley (1969) suggests the p o s s i b i l i t y of r e c i p r o c a l induction by mutual transfer of substances, such as amino acids and proteins, between the germinal e p i t h e l i a l c e l l s and underlying f o l l i c l e c e l l s i n early development. 5 Accordingly, i n the present study, exogenous electron microscopic tracers were chosen i n an attempt to examine and characterize the germinal epithelium i n terms of i t s active and/or passive transport properties. B. Electron Microscopic Tracers The use of electron microscopically demonstrable tracers to elucidate pathways of normal molecular uptake, transfer, and release from c e l l s has become of increasing value i n recent years. Currently used tracers represent a considerable range of molecular size and weight, and as such constitute a graded series of molecular probes with which c e l l s and tissues may be investigated. In both q u a l i t a t i v e and quantitative 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 of a tracer depends also on a number of other important factors. F i r s t l y , the dosage required should not be i n i m i c a l to the continued normal p h y s i o l o g i c a l functioning of the animal. Also, molecules of the tracer 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 of r e s u l t s . In addition, the tracer should not be metabolized by the tissues nor should i t be r a p i d l y cleared from the body. Electron microscopic tracers may be c l a s s i f i e d into two main groups. The f i r s t consists of molecules which are detect- able by v i r t u e of t h e i r natural electron opacity. Once having exposed tissues to t h i s type of tracer, routine processing and examination w i l l reveal the presence (or absence) of these molecules. Tracers of this type may be of either b i o l o g i c a l or non-biological o r i g i n . They are exemplified by lanthanum (Revel and Karnovsky, 1967), carbon (Leak, 1971). latex spheres (Leak, 1971). f e r r i t i n (see p. 8 ), dextrans (Simionescu and Palade, 1971). glycogen (Simionescu and Palade, 1971). and c o l l o i d a l suspensions of gold (Ghiquoine, 1961) and mercuric sulphide (Odor, 1956) . The second group of tracers i s enzymatic i n nature. The s i t e of t h e i r l o c a l i z a t i o n within a tissue or c e l l i s v i s i b l e only subsequent to the exposure of the tissue to a substrate appropriate to the p a r t i c u l a r enzyme used as a tracer. What i s then seen i s the electron microscopically v i s i b l e end product of the reaction, which should be l o c a l i z e d as a prec i p i t a t e within the immediate v i c i n i t y of the enzyme. Tracer molecules of the second type must be by d e f i n i t i o n of b i o l o g i c a l o r i g i n , and are mainly peroxidatic enzymes 1 horseradish peroxidase (HRP) (see p. 8 ), myeloperoxidase (Graham and Karnovsky, 1966a), lactoperoxidase (Graham and Kellermeyer, 1968), microperoxidase (Feder, 1970), catalase (Goodenough and Revel, 1971). myoglobin (Anderson, W.A., 1972b), and cytochrome c (Karnovsky and Rice, I969). Experimentally the s i t e s of l o c a l i z a t i o n of these enzymes within a tissue 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 reaction involving hydrogen peroxide and 3,3'-diaminobenzidine (DAB) (Graham and Karnovsky, 1966b; H i r a i , 1975). As an example the reaction sequence of HRP with H 20 2 and DAB i s shown below (modified a f t e r White et a l . , 1973). To show that DAB i s i n i t i a l l y a hydrogen donor i n the following reaction, i t i s represented as DABHg. HRP-H20 + H 20 2 >- HRP-H202 + HgO (complex I) 7 HRP-H202 + DABH2 >- complex II + DABH • complex II + DABH - >• HRP-HgO + DAB The reaction sequence involves the formation of consecutive complexes which have not yet been pr e c i s e l y defined. The over- a l l r e s u l t of the reaction i s the regeneration of HRP, which w i l l continue to catalyze the same reaction, and the formation of oxidized DAB, which forms an insoluble p r e c i p i t a t e at the s i t e s of reaction (Seligman et a l . , 1968). The p r e c i p i t a t e is= a brown pigment at the l i g h t microscopic l e v e l and an electron opaque substance at the electron microscopic l e v e l . The aforementioned c r i t e r i a f o r choosing tracers, as well as the successful experience of other workers with c e r t a i n of these tracers, were taken into consideration when choosing tracer molecules with which to investigate the germinal epithelium. I t was decided to use two tracer molecules, f e r r i t i n and HRP. By v i r t u e of t h e i r respective molecular dimensions and weight they would be expected to have d i f f e r e n t transfer c h a r a c t e r i s t i c s , and thus bound a sizeable range of possible tracers which could be used. F e r r i t i n , the larger of the two tracers, i s a heme protein containing 20-24% i r o n which was f i r s t i s o l a t e d from horse spleen (Ainsworth and Karnovsky, 1971). The molecule consists of a s p h e r i c a l s h e l l of protein surrounding a core of f e r r i c o hydroxide micelles. The t o t a l molecular diameter i s 110 A, o whereas the core diameter i s approximately 55 A (Ainsworth and Karnovsky, 1971). The core i s the electron dense part of the molecule. In order to avoid misinterpretation of results i t 8 must therefore be kept i n mind that the entire molecule i s not seen i n electron micrographs. The molecular weight of f e r r i t i n i s about 462,000 (Ainsworth and Karnovsky, 1971). The second tracer, HRP, has also been extensively used i n tracer studies. The reason f o r the e f f i c a c y of t h i s tracer i s aptly described by Graham and Karnovsky (1966b)t "the method i s sensitive because enzymatic a c t i v i t y has an ampli- f y i n g e f f e c t j thus a few molecules of protein at a s i t e can generate a much larger amount of reaction product upon incuba- o t i o n . " HRP has a molecular diameter of about 40 A and a molecular weight of about 40,000 (Klapper and Hackett, 1965). Commercially available HRP contains a number of components which are separable by starch electrophoresis. The amino a c i d composition, absorption spectrum, s i z e , and enzymatic a c t i v i t y of these components are a l l quite 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 fracti 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 that the t o t a l enzymatic a c t i v i t y of unfractionated HRP would not be affected by d i f f e r e n t r e l a t i v e amounts of the f r a c t i o n s . HRP and f e r r i t i n have been used as tracers 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 to study the permeability of c a p i l l a r i e s i n cardiac and s k e l e t a l muscle (Karnovsky and Cotran, 19665 Karnovsky, 1967? Bruns and Palade, 1968), lung (Schneeberger- Keeley and Karnovsky, 19685 Clementi, 1970), inte s t i n e (Clementi and Palade, 1969). cerebrum (Reese and Karnovsky, 1967), 9 thymus (Gervin and Holtzman, 1972), ovary (Anderson, W., 1972a; Payer, 1975). and renal glomerulus (Farquhar and Palade, 1961; Farquhar et a l . , 1961; Webber and Blackbourn, 1970). They have also been used to study permeability and absorption i n proximal rena l tubule (Graham and Karnovsky, 1966b; Maunsbach, 1966), mesothelium (Karnovsky and Cotran, 1966; Cotran and Karnovsky, 1968; Kluge, 1969). pericardium (Kluge and Hovig, 1968; Kluge, 1969). ovarian f o l l i c l e (Anderson, W., 1972a; Payer, 1975). oocyte (Anderson, S., 1967. 1972), p a r i e t a l layer of Bowman's capsule (Webber and Blackbourn, 1971). post-ovulatory zona p e l l u c i d a (Hastings et a l . , 1972), urinary bladder epithelium (Wade and Discala, 1971), and cultured and normal tumor c e l l s (Ryser et a l . , 1962). F e r r i t i n can also be linked, using b i f u n c t i o n a l conjugating agents, to antibody, i n order to l o c a l i z e s i t e s of antigen- antibody reactions. Protein compounds l a b e l l e d with f e r r i t i n have included antifibrinogen (Wylie, 1964), enzymes (Benjaminson et a l . , 1966), and a n t i v i r a l globulin (Morgan et a l . , 1961). G. Scope of the Present Study A description of the normal ultrastrueture of pre- and postpubertal r a t germinal epithelium w i l l be presented, as the epithelium of t h i s species has not yet been adequately described i n the l i t e r a t u r e i n terms of i t s fine structure. The present study i s also concerned with the movement of two tracers, HRP and f e r r i t i n , from the peritoneal cavity into and across the germinal epithelium. By observing the l o c a l i z a t i o n o f these t r a c e r s with the e l e c t r o n microscope, i t was a n t i c i - p ated t h a t the a c t i v e and/or p a s s i v e t r a n s p o r t c a p a c i t i e s o f the germinal e p i t h e l i u m might be e l u c i d a t e d . 11 MATERIALS AND METHODS Female albino rats 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 representing both pre- and postpubertal animals. Food (Purina Rat Chow) and water were provided ad libitum. Adult animals were selected without regard to the exact stage of the ovarian cycle. HRP (type I I , Sigma Chemical Company, St. Louis, Missouri) and f e r r i t i n (horse spleen, 2X c r y s t a l l i n e , cadmium free, N u t r i t i o n a l Biochemical Corporation, Cleveland, Ohio, or ICN Pharmaceuticals, Inc., L i f e Sciences Group, Cleveland, Ohio) were chosen as the electron microscopic tracers. HRP was used i n dosages varying from 8-100 mg/100 g body weight and i n concentrations ranging from 10-40 mg/ml isotonic s a l i n e . F e r r i t i n dosages of 20-200 mg/100 g body weight, at concentra- tions of 10-100 mg/ml isotonic s a l i n e , were used. Animals received 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 of one or other of the tracers i n the r i g h t lower quadrant of the abdomen. After s p e c i f i c periods of time these animals were anesthetized and s a c r i f i c e d . In addition, some animals were anesthetized and t h e i r ovaries d i r e c t l y immersed i n f e r r i t i n at a concentration of 100 mg/ml isotonic s a l i n e . In either case the anesthetic consisted of a sequential combination of i n t r a p e r i t o n e a l l y injected sodium pentobarbital (concentration! 3-3$s dosage i 0.2 ml/100 g) followed by subcutaneously injected sodium phenobarbital (concentration! Z,0%i dosagei 0.2 ml/100 g). Animals i n j e c t e d with HRP were s a c r i f i c e d at 45 min, 65 min, 12 2 hr, 4 hr, and 5 hr. Post-injection 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 njections were 30 min, 45 min, 1 hr, 2 hr, 4 hr, 4§ hr, and 24 hr. Post-immersion s a c r i f i c e times f o r anesthetized animals with t h e i r ovaries immersed i n f e r r i t i n s olution were 15 min, 30 min, 1 hr, l i hr, 2 hr, and 3* hr. To expose the ovaries, a midline i n c i s i o n was made through the ventral abdominal wall into the peritoneal cavity from a po s i t i o n just r o s t r a l to the vaginal opening as f a r as the subcostal l i n e . Fixative was immediately introduced into the peritoneal c a v i t y at this point i n those animals which had received p r i o r injections of tracer. Lateral i n c i s i o n s were then made fo r approximately 1 cm on either side of the caudal end of the f i r s t i n c i s i o n . The abdominal wall flaps so created were then retracted and most of the small i n t e s t i n e displaced to the outside of the abdominal cavity. This manoeuvre exposed the ovaries. Each was situated near the end of each horn of the uterus, separated from i t by the highly c o i l e d oviduct. A tendinous band, t r a v e l l i n g within the broad ligament from the dorsal aspect of each ovary to a point on the dorsal body wall adjacent to the i p s i l a t e r a l diaphragmatic crus, maintained each ovary i n pos i t i o n . This band was cut and the uterine horn, oviduct, and ovary r a i s e d o f f the dorsal body wall so that the ovary, contained within i t s bursa, could be removed. The r e l a t i o n s h i p of the oviducal mesenteries to the mammalian ovary i s highly variable (Beck, 1972). There may be complete anatomic independence of these structures, with much of the 13 surface 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 closed bursa derived from the oviducal mesenteries, such as that seen i n the golden hamster (Clewe, 1966). The s i t u a t i o n that prevails i n the r a t resembles most c l o s e l y that seen i n the golden hamster. The important difference, however, from the point of view of i n t r a p e r i t o n e a l tracer introduction, i s that the periovarian bursa of the r a t has on i t s ventromedial side a small opening by which the peritoneal cavity and the periovarian space may communicate. Due to t h i s d i r e c t communication i t was not necessary to s u r g i c a l l y remove or r e t r a c t the bursa i n order to expose the ovarian e p i t h e l i a l surface more f u l l y . Even though t h i s procedure might have expedited tracer movement, i t would have added unnecessary s u r g i c a l complications to the procedure. These complications could be avoided by increasing the time of exposure to tracer, which would overcome the r e s t r i c t i o n on bulk f l u i d movement which a single small bursal opening would be expected to pose. Immediately a f t e r severing i t s attachments the encapsulated ovary was removed and placed i n f i x a t i v e . The bursa was then c a r e f u l l y dissected away to promote a more rap i d f i x a t i o n of the germinal epithelium. After preliminary experiments, i n t r a - vascular perfusion f i x a t i o n was deemed to be of no added advantage i n preservation of germinal e p i t h e l i a l c e l l u l t r a - structure. Like a l l e p i t h e l i a , the germinal epithelium i s not vascularized. Thus i t would not be subject to the action of 14 the f i x a t i v e by d i f f u s i o n from blood vessels any sooner than i t would be with immersion f i x a t i o n . In those anesthetized animals i n which the ovaries were immersed i n f e r r i t i n solution, the basic s u r g i c a l procedure was s i m i l a r to that just described. However, the period of time between exposing the ovaries and s a c r i f i c i n g the animal was extended to correspond to s a c r i f i c e times of animals re c e i v i n g i n t r a p e r i t o n e a l tracer i n j e c t i o n s . After d i v i d i n g the dorsal tendinous ligaments, adipose tissue surrounding each ovary and i t s adnexa was teased away. Each uterine horn, together with i t s attached oviduct and ovary, was raised so that the ovary could be placed i n a small p l a s t i c container f i l l e d with f e r r i t i n s o lution (the container was intra-abdominally situated). The periovarian bursa was removed i n some animals p r i o r to t h i s step. The small intestine was repositioned within the abdominal cavity and the cut edges of the i n c i s i o n approximated. After the desired period of immersion the f e r r i t i n s o l u t i o n was d i s - placed by f i x a t i v e . After b r i e f i n s i t u f i x a t i o n the ovaries were removed and placed i n fresh f i x a t i v e for a further period of time. After removal of the ovaries, 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 allowing the animals to exsanguinate. The f i x a t i v e used i n t h i s study consisted of a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde i n 0.1 M cacodylate buffer adjusted to pH 7.3 (a l i l d i l u t i o n of the f i x a t i v e described by Graham and Karnosvsky, 1966a). Ovaries were 15 f i x e d f o r 3-20 hr at room temperature (20° C). After f i x a t i o n , the procedures used for processing tissues f o r l i g h t and trans- mission electron microscopy diverged from those used for pre- paring specimens f o r scanning electron microscopy. For l i g h t and electron microscopic studies, f i x e d ovaries were embedded i n 7% agar, sectioned on a S o r v a l l TC-2 tissue chopper at 200 jam, rinsed several times i n co l d 0.1 M cacodylate buffer, and stored i n buffer at 4° C f o r 1-48 hr. Sections from ovaries which had been exposed to HRP were then transferred from buffer to an incubation s o l u t i o n which contained the substrate f o r HRP. The s o l u t i o n consisted of 0.05% 3,3 ,-diaminobenzidine tetrahydrochloride i n 0.05 M t r i s buffer containing 0.01# hydrogen peroxide at pH 7.6 (Graham and Karnovsky, 1966b). The sections were exposed f o r 40 min at room temperature and were then again rinsed i n 0.1 M cacodylate buffer. A l l sections were then post-fixed i n buffered 1% osmium tetroxide f o r 1 hr, rinsed i n d i s t i l l e d water, and stained en bloc i n a saturated aqueous solution of uranyl acetate f o r 1 hr. Tissues were then dehydrated through an ascending ethanol series and propylene oxide, i n f i l t r a t e d i n vacuo, and embedded i n a l i l mixture of epon-araldite polymerized at 60° C. Thick (0.5jum) and t h i n (silver-grey) sections were cut on an LKB Ultrotorae I I I , using either glass or diamond knives. Thick sections were stained with 1% aqueous to l u i d i n e blue and were used f o r preliminary l i g h t microscopic i d e n t i f i c a t i o n and orie n t a t i o n of areas of i n t e r e s t . Thin sections were mounted 16 on uncoated copper grids and examined i n a P h i l i p s 200 or 300 electron microscope. A l l t h i n sections were f i r s t examined without a d d i t i o n a l membrane sta i n i n g to avoid misinterpretation of r e s u l t s due to possible lead c i t r a t e s t a i n i n g artefacts. In the case of sections not stained with lead c i t r a t e , the operating voltage of the microscope was reduced from 60 kV to 40 kV to enhance contrast. For scanning electron microscopic studies the following procedure was used. After i n i t i a l f i x a t i o n , ovaries were rinsed i n several changes of cold 0.1 M cacodylate buffer. After being stored i n buffer at k° C f o r 1-48 hr, ovaries were post-fixed f o r 1 hr i n buffered 1% osmium tetroxide. They were then rinsed i n d i s t i l l e d water and dehydrated through an ascending ethanol s e r i e s . The ethanol was then substituted by iso-amyl acetate by processing the tissues through an ethanol-iso-amyl acetate series of increasing iso-amyl acetate concentration. Iso-amyl acetate i s a polar solvent miscible with carbon dioxide, 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 point drying procedure. Carbon dioxide prevents ice c r y s t a l formation during the procedure. Ovaries were c r i t i c a l point dried, mounted on aluminum stubs, and coated with gold i n a vacuum evaporator. Specimens were examined i n a Cambridge Stereoscan Model S4 microscope. Control animals, representing the same age groups as the experimental animals, were also prepared f o r transmission and scanning electron microscopic examination by the methods just described. Control animals received e i t h e r one of two treatments. 17 Some animals received no intraperitoneal i n j e c t i o n corresponding to a tracer i n j e c t i o n . They were anesthetized and t h e i r ovaries removed and processed f o r examination. This would be expected to show the normal structure of the germinal epithelium and reveal any endogenous f e r r i t i n or f e r r i t i n - l i k e molecules. As well, when reacted i n the incubation medium, any endogenous peroxidase a c t i v i t y would be shown. By comparison with the experimental r e s u l t s , any changes due to the tracer i n j e c t i o n by v i r t u e of either i t s volume or i t s composition would also be revealed. The second group of animals received an intraperitoneal i n j e c t i o n of isotonic saline of a volume s i m i l a r to that of tracer administered to an animal of the same weight. After a period corresponding to the s a c r i f i c e time of an animal receiving a tracer i n j e c t i o n , the ovaries of the control animal were processed. In this way any changes due to the saline could be detected. Any other changes seen i n the germinal epithelium could then be a t t r i b u t e d to the tracer i t s e l f or to the saline and tracer i n combination with one another. As i n the f i r s t c o n t r o l group, some of the ovarian tissue was also reacted f o r endogenous peroxidase a c t i v i t y . I - 18 RESULTS A. Normal Germinal Epithelium The following r e s u l t s apply to a l l animals, both pre- and postpubertal, receiving either no intraperitoneal i n j e c t i o n (other than anesthetic) or an i n j e c t i o n of saline alone. 1. General E p i t h e l i a l Morphology As seen with the l i g h t and transmission electron micro- scopes, ovarian germinal epithelium i n section ranges from a simple squamous to a simple cuboidal type, with a l l possible intermediate variations ( f i g . 1). This f i n d i n g was corroborated and extended by scanning electron microscopic studies which showed overall surface morphology of the germinal e p i t h e l i a l c e l l s as well as the regional d i s t r i b u t i o n of c e l l shape. This correlated with the degree of maturation of underlying f o l l i c l e s ; (see Discussion). Cuboidal c e l l s are found i n the "va l l e y s " or crypts between f o l l i c l e s . The cuboidal c e l l s give way i n a graded fashion to squamous c e l l s overlying the f o l l i c l e s ( f i g . 2). Subsequent examination of sectioned material showed that no c y t o l o g i c a l differences other than shape distinguish these two c e l l types. Typical examples of cuboidal and squamous c e l l s are shown i n figures 3 and 4. In a l l specimens examined, the germinal e p i t h e l i a l c e l l s seem to represent a continuous c e l l u l a r covering f o r the ovary. Occasionally, i n scanning electron micrographs, s i t e s of apparent i n d i v i d u a l c e l l necrosis are seen ( f i g . 2). In contrast, more 19 extensive areas of denudation of e p i t h e l i a l c e l l s have been observed i n studies of the human ovary (Papadaki and Beilby, 1971). The e p i t h e l i a l c e l l s l i e on a basement membrane of variable thickness ( f i g s . 1,3,4). They are separated from the region of the developing f o l l i c l e s by the tunica albuginea o v a r i i i n both pre- and postpubertal animals. No continuity 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 imperceptibly 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 i n t e r s t i t i a l matrix of the tunica albuginea o v a r i i . Collagen f i b r e s are randomly scattered throughout the basement membrane but seem to be more abundant i n i t s deeper regions ( 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 i n t e r c e l l u l a r c l e f t s and junc- tions . The most s t r i k i n g feature of the i n t e r c e l l u l a r c l e f t s of the germinal epithelium i s the extreme ' v a r i a b i l i t y of t h e i r course ( f i g s . 1,3,4). This v a r i a b i l i t y i s a function of the i n t e r d i g i t a t i o n of cytoplasmic processes of i r r e g u l a r s i z e and shape from adjacent c e l l s . There i s no common pattern f o r a l l i n t e r c e l l u l a r c l e f t s . A r e l a t i v e l y short and d i r e c t c l e f t is the exception, and usually the c l e f t s are tortuous, convoluted, or even labyrinthine i n t h e i r complexity ( f i g . 5). Many of the c e l l s may overlap one another for considerable distances. The most obvious way i n which germinal e p i t h e l i a l c e l l s are adherent i s v i a the i n t e r d i g i t a t i n g processes just mentioned. 20 This i s a purely mechanical interl o c k i n g . Such union does not require the maintenance of a constant i n t e r c e l l u l a r distance f o r i t s function, as do some more s p e c i a l i z e d types of junctions. Nonethelesss, membranes often p a r a l l e l one another quite c l o s e l y along the path of a c l e f t , regardless of i t s complexity. Thus a very tortuous c l e f t may display a f a i r l y regular 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 along the basal h a l f of t h e i r course, membranes are often greatly separated from each other. These regions sometimes look merely l i k e large gaps. Usually, however, the appearance i s suggestive of large polymorphic vacuoles either fusing with or budding o f f one or both of the membranes bounding the c l e f t ( f i g s . 1,3). The density of the contents of these "vacuoles" i s i d e n t i c a l to that of the i n t e r c e l l u l a r substance of the more c l o s e l y approximated regions of the c l e f t s . Therefore, these areas probably represent sections through large spaces which e x i s t due to i r r e g u l a r i t i e s i n the shape and pattern of in t e r l o c k i n g cytoplasmic processes. Pinocytotic invaginations and ve s i c l e s appear quite often along the course of the membranes bounding the i n t e r c e l l u l a r c l e f t s . These w i l l be discussed further i n section 4, under Organellar structure. Small punctate regions or f o c i of very close membrane approximation, and i n some cases fusion, are frequently seen along some of the c l e f t s ( f i g . 6). Their appearance i n section suggests that they may represent macular forms of gap junctions or occluding junctions, or sections of zonular junctions of the same types. Often there i s an increase i n the cytoplasmic 21 density subjacent to this f o c a l type of junction. There are often multiple randomly scattered f o c i along a c l e f t , t h e i r numbers usually being greater near the peritoneal end. Less numerous are extended versions of the f o c a l junctions just described, which s i m i l a r l y may be multiple along a single c l e f t ( f i g . ?). Again there i s a d i f f i c u l t y i n ascertaining whether adjacent membranes are just extremely close or actually fused. Occasionally, long regions of apparent membrane fusion 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 resolved to actually be composed of two d i s t i n c t c e l l membranes separated by a small but d e f i n i t e 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 surface modifications appear mainly as micro- v i l l i and pinocytotic 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 quite random from c e l l to c e l l and are best appreciated with scanning electron micrographs of the c e l l free surface ( f i g . 10). In section the m i c r o v i l l i are seen to contain ground cytoplasm and an i n t e r n a l skeleton of microfilaments p a r a l l e l to the long axis. Pinocytotic invaginations are seen to open on to the free surface i n both transmission and scanning electron micrographs ( f i g s . 3i^ill)« The s i z e of the invaginations i s variable, as i s t h e i r d i s t r i - bution. Irregular cytoplasmic evaginations of the free surface, as described i n other species (Gondos, 1969; Papadaki and Beilby, 1971), are sometimes seen. C i l i a are seen only infrequently, 22 even i n scanning electron microscopic studies, c. Basal c e l l membrane. The contour of the c e l l basal surface varies from c e l l to c e l l , being r e l a t i v e l y s t r a i g h t i n some and highly i r r e g u l a r i n others. Pinocytotic ve s i c l e s open on to t h i s surface, although they are fewer i n number than at the free surface. There are no junctional s p e c i a l i z a t i o n s along the basal membrane. 3. Nuclear Structure Germinal e p i t h e l i a l c e l l nuclear shape r e f l e c t s that of the c e l l ( f i g s . 3,4). Nuclei have o v e r a l l shapes i n section ranging from f l a t and elongated to almost c i r c u l a r . The r e l a t i v e proportion of the c e l l occupied by the nucleus varies with the plane of section. The nuclear contour i s usually i r r e g u l a r ( f i g s . 1,4). Cytoplasmic projections of varying dimensions extend into the nucleus ( f i g . 1), and sometimes cause the nucleus to assume a pseudolobulated form. Mitochondria and v e s i c l e s • are sometimes seen i n these extensions. The nucleus i s deline- ated by the usual bilameHate nuclear envelope, along which nuclear pores are found. Some of these pores are closed by a th i n diaphragm. Ribosomes are adherent to the outer lamella of the envelope ( f i g . ?). Most of the chromatin i s circ u m f e r e n t i a l l y d i s t r i b u t e d against the inner aspect of the nuclear envelope, with some located as coarse clumps throughout the nucleus (figs. 1,3,4). A l l n u c l e i examined were i n the interphase state. Nucleoli are rar e l y cut i n section ( f i g . 4). 23 4. Organellar Structure Rough endoplasmic reticulum i s found i n variable amounts throughout the germinal e p i t h e l i a l c e l l cytoplasm. I t i s usually c l o s e l y associated with mitochondria, with which i t may form large aggregates ( f i g s . 1,3,4,7). The contents of the cisternae appear to be of about the same density as the cyto- plasm. The degree of d i l a t i o n of the cisternae varies both within a c e l l and from c e l l to c e l l . Smooth endoplasmic reticulum i s very seldom seen. Both the mitochondria rel a t e d to the rough endoplasmic i • reticulum and those d i s t r i b u t e d randomly throughout the cyto- plasm have a s i m i l a r appearance. The matrix i s denser than the surrounding cytoplasm and the contents of most v e s i c l e s , and i n section mitochondrial shape varies from c i r c u l a r to ovoid to elongate ( f i g s . 3,4). Cristae appear as flattened lamellae whose orientation within the mitochondria i s not s p e c i f i c . The Golgi complex consists of a variable number of lamellae of d i f f e r e n t shape and s i z e . Usually only one Golgi complex i s observed i n each c e l l . I t i s situated i n a l a t e r a l or superior paranuclear p o s i t i o n i n most instances. I t i s not observed between the nucleus and the basal c e l l surface. Vesicles presumably derived from the Golgi complex are seen about i t s entire perimeter and are not p r e f e r e n t i a l l y r e l a t e d to any , p a r t i c u l a r aspect of the complex ( f i g . 4 i n s e t ) . The degree of f i l l i n g and electron density of the contents of these vesic l e s i s variable. Ribosomes are very numerous. As well as being c l o s e l y 24 associated with the endoplasmic reticulum and nuclear envelope, they are observed i n large numbers free i n the cytoplasm and occasionally i n polyribosomal groupings. Cytoplasmic v e s i c l e s are seen throughout the c e l l and at a l l c e l l surfaces. They are of highly diverse s i z e and shape. Many of the vesicl e s are near the a p i c a l and l a t e r a l c e l l sur- faces and may be derived from pinocytotic a c t i v i t y . They could also be pinocytotic invaginations sectioned i n a plane other than that i n which t h e i r opening to a surface can be seen. The amount and d i s t r i b u t i o n of electron dense material within a ve s i c l e ranges considerably, as previously noted f o r Golgi v e s i c l e s . Some v e s i c l e s contain what appear to be smaller ve s i c l e s inside them, thus forming multivesicular bodies ( f i g . 4 i n s e t ) . Somewhat denser structures s i m i l a r to these have been i d e n t i f i e d as autophagic vacuoles i n human germinal epithelium (Papadaki and Beilby, 1971). Cytoplasmic inclusions which may represent l i p i d stores are seen i n some c e l l s ( f i g s . 3,4). Their s i z e and d i s t r i b u t i o n within a germinal e p i t h e l i a l c e l l i s not constant and i n section usually no more than two or three are ever noted within a c e l l . The subjacent stromal and granulosa c e l l s usually contain more l i p i d inclusions than the e p i t h e l i a l c e l l s . Microfilaments are seen i n the cytoplasm of some germinal e p i t h e l i a l c e l l s ; usually 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 is 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 in the ovary within 45 min of exposure to tracer, they differ markedly in their depth of penetration. HRP is found not only in the germinal epithelium but also deep within the tunica albuginea ovarii within this period of time. Fe r r i t i n on the other hand appears only in germinal epithelial cells at the end of 45 min, and requires several hours before i t appears in the cells and interstitium of the tunica albuginea ovarii. Endogenous peroxidase activity is limited to the cytoplasm of erythrocytes within ovarian blood vessels. No electron dense particles 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 in an electron micrograph. Rather i t is the electron dense enzymatic reaction product which is visualized 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 is further enhanced after i t s reaction with osmium tetroxide during post-fixation (Graham and Karnovsky, 1966b). HRP is observed on the germinal epithelial c e l l apical surface, in the intercellular clefts, in pinocytotic invagina- 26 tions, at a l l e p i t h e l i a l (and some fi b r o b l a s t ) c e l l surfaces, i n v e s i c l e s and multivesicular bodies of 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 , i n the basement membrane, and i n the i n t e r s t i t i u m of the tunica albuginea o v a r i i ( f i g . 12). The pattern of tracer uptake was s i m i l a r i n a l l experiments. HRP i s observed to be most abundant i n the e x t r a c e l l u l a r regions of the ovary. There i s a marked e x t r a c e l l u l a r gradient of HRP density from the e p i t h e l i a l free surface 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 extent of the gradient varies with the time of exposure to HRP. Collagen f i b r e s are especially well 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 to the negative s t a i n - ing e f f e c t of HRP. Pinocytotic invaginations containing variable amounts of HRP are seen at a l l surfaces of the germinal e p i t h e l i a l c e l 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 surfaces. Such invagina- tions are also seen, but much less frequently, at the e p i t h e l i a l c e l l basal surface and at the surface of f i b r o b l a s t s . Occasion- a l l y HRP was not seen at the a p i c a l surface, even though i t appeared elsewhere throughout the epithelium and subjacent tissues ( f i g . 14). This l o c a l removal of HRP was probably attributable to excessive washing of tissue i n buffer p r i o r to pos t - f i x a t i o n , and has been noted i n other studies:where HRP has been used as a tracer (Cotran and Karnovsky, 1968). Vesicles containing HRP are found throughout germinal e p i t h e l i a l c e l l s . They constitute a variable but reasonably small proportion of the t o t a l number of c e l l vesicles. Even af t e r exposure to HRP for 5 hrs, the proportion of tracer- containing v e s i c l e s was s t i l l quite small. Some vesicles are completely f i l l e d with the tracer, which appears either to be amorphous or to have a coarse granular appearance. In other v e s i c l e s which are not completely f i l l e d with HRP, the tracer appears as small clumps s i m i l a r to those often seen at the c e l l a p i c a l surface, or as an inner circumferential coating. Vesicles containing HRP are sometimes seen i n f i b r o b l a s t s ( f i g . 12), but not i n c a p i l l a r y endothelial c e l l s . Peripherally, 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 surfaces than the basal surface. Apical surface v e s i c l e s are of a f a i r l y uniform size and contour, as are those at the basal surface. Vesicles near the l a t e r a l c e l l surface may either be s i m i l a r to those seen at the other c e l l surfaces, or they may be quite variable i n size and shape. The d i s t r i b u t i o n of v e s i c l e s at the a p i c a l and l a t e r a l surfaces p a r a l l e l s that of the pinocytotic invaginations described e a r l i e r . After excessive buffer washing the proportion of a p i c a l surface v e s i c l e s containing HRP i s greatly reduced ( f i g . 14). This would not be expected of true v e s i c l e s and suggests that many so-called " v e s i c l e s " devoid of tracer subsequent to excessive washing are i n f a c t pinocytotic invaginations continuous with the c e l l a p i c a l surface i n another plane. There are, however, other tracer-containing v e s i c l e s within the c e l l s whose contents are not removed by prolonged washing ( f i g . 12). Of these v e s i c l e s some are quite close to the l a t e r a l c e l l surfaces 28 bounding the i n t e r c e l l u l a r c l e f t s . Certain of these could be true v e s i c l e s derived from the l a t e r a l c e l l surface, whereas others could be invaginations s i m i l a r to those seen at the a p i c a l surface. In either case they could have been f i l l e d with tracer 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 that they could also be v e s i c l e s from the a p i c a l surface emptying t h e i r contents into the i n t e r c e l l u l a r c l e f t s . 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 of cyto- 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 to the appearance of large invaginations into the c e l l from the l a t e r a l c e l l surfaces. Sectioning through such an invagination i n a plane other than that 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 variable contour. Some of these "vesicles"may be quite removed from the l a t e r a l c e l l surface, depending on the length of the invagination. As seen i n f i g . 15t these invaginations can be very long. In t h i s p a r t i c u l a r case, sectioning i n another plane could e a s i l y r e s u l t i n the creation of an apparent v e s i c l e deep within the c e l l . On occasion the appearance i s also created of large t r a c e r - f i l l e d v e s i c l e s to which are connected smaller v e s i c l e s , These smaller v e s i c l e s seem to be either forming from,or emptying t h e i r contents into, the large v e s i c l e ( f i g . 15 i n s e t ) . However, such large v e s i c l e s are always situated near i n t e r c e l l u l a r c l e f t s , never deep within a c e l l . They are also seen i n control material. 29 There s t i l l remain a number of t r a c e r - f i l l e d v e s i c l e s within the c e l l which are so situated that i t would be d i f f i c u l t to envisage t h e i r connection with any of the c e l l surfaces. They are on average smaller and more regular i n outline than would be expected of "vesicles'* derived from section of l a t e r a l surface invaginations. One would have to invoke the existence of very long and numerous surface membrane invagina- tions of small and regular diameter penetrating deeply into the c e l l which, when cut i n cross-section, would appear as v e s i c l e s . I f t h i s were the case, then oblique and longitudinal sections of such channels should be seen i n some micrographs, and they are not. To completely eliminate t h i s p o s s i b i l i t y s e r i a l sections were examined. This demonstrated the independence of these v e s i c l e s from any c e l l surface. There are thus some tra c e r - f i l l e d v e s i c l e s which would seem to constitute 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, suggesting that i f tracer transport does occur, 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 basal surface or occurs to only a minor extent i n that d i r e c t i o n . I t i s not possible to t e l l whether or not such vesi c l e s a c t i v e l y transport tracer from the c e l l a p i c a l surface or to or from the c e l l l a t e r a l surfaces. Within the i n t e r c e l l u l a r c l e f t s HRP may be reasonably homogeneous ( f i g s . 16,1?), or i t may present a granular appear- ance ( f i g . 18). The presence of the tracer within the c l e f t s emphasizes t h e i r t o r t u o s i t y . The continuity of HRP from the c e l l free surface to the basement membrane varies within d i f f e r e n t 30 c l e f t s . I t seems to be a function of the presence of junctions of the types mentioned e a r l i e r (compare f i g s , 16,1?,18). Unfortunately, the,presence of HRP within a c l e f t often seems to obscure the l a t e r a l membrane surfaces. Thus junctional 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 discern and interpret along 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 junctions or extended versions thereof can be seen, HRP seems to be found on e i t h e r side of the point of close c e l l approximation. I t cannot be distinguished whether HRP i s present within the junctional area i t s e l f or whether i t has diffused around the junction to appear on both sides of i t ( f i g . 17). When two or more f o c a l junctions are seen along a c l e f t , the region between adjacent junctions i s sometimes devoid of tracer ( f i g , 18). This suggests that some junctions are d e f i n i t e l y not permeable to HRP. I t also suggests that these p a r t i c u l a r junctions are not simple maculae occludentes, otherwise HRP could have diffused around them to f i l l the intervening space, These junctions may perhaps represent cross sections through an annular junction between adjacent l a t e r a l c e l l surfaces, Such a l o c a l r i n g of occlusion would prevent HRP from being l o c a l i z e d i n i t s i n t e r i o r and would lead to the appearance of a region such as shown i n f i g . 18. Again, freeze-fracture studies would probably be the most suitable 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 of the junctions of the germinal epithelium. 2. F e r r i t i n D i s t r i b u t i o n F e r r i t i n l o c a l i z a t i o n i n ovaries of animals injected with 31 f e r r i t i n and i n animals whose ovaries were d i r e c t l y immersed in f e r r i t i n i s e s s e n t i a l l y s i m i l a r . I t i s found i n t r a - and e x t r a c e l l u l a r l y , with the notable exception that 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 germinal epithelium. Subsequent to f e r r i t i n exposure, a variable number of germinal e p i t h e l i a l c e l l s are seen to contain f e r r i t i n within iriembrane-bounded v e s i c l e s and multivesicular bodies ( f i g . 19) • F e r r i t i n molecules are not usually observed i n e p i t h e l i a l i n t e r c e l l u l a r c l e f t s (see Discussion). Some f i b r o b l a s t s of the tunica albuginea ( f i g . 20) and some c a p i l l a r y endothelial c e l l s ( f i g . 21) also contain f e r r i t i n within v e s i c l e s . A possible sequence of the pinocytotic events underlying f e r r i t i n uptake i s shown i n f i g . 22. I t i s r e a l i z e d that the v e s i c l e containing f e r r i t i n i n f i g . 22 could a c t u a l l y be an invagination sectioned i n a plane i n which i t i s not connected to the surface. Nevertheless, as f e r r i t i n i s found deep to the germinal epithelium and i s probably not transported through the i n t e r c e l l u l a r c l e f t s (see Discussion), i t i s assumed that the sequence shown i n f i g . 22 a c t u a l l y mirrors 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 . Occasionally, 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 to contain free 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 c e l l v e s i c l e s , but not i n the zona p e l l u c i d a or i n oocytes. Blood plasma within c a p i l l a r i e s and venules of the tunica albuginea ( f i g . 21) and f o l l i c u l a r f l u i d ( f i g , 23) are frequently seen to contain free f e r r i t i n molecules. F e r r i t i n molecules are always seen to be discrete p a r t i c l e s . They do not form the type of amorphous clumps 32 c h a r a c t e r i s t i c of HRP. Both the number and d i s t r i b u t i o n of 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 within a c e l l and 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 within a single v e s i c l e exhibit wide v a r i a - t i o n . In spite of t h e i r variable 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 represent more than a r e l a t i v e l y small propor- t i o n of a l l c e l l v e s i c l e s . The number of f e r r i t i n molecules within a v e s i c l e ranges from as few as one to as many as several thousand, and both extremes may occur i n a single c e l l . Vesicles with the highest f e r r i t i n concentration are found i n the f i b r o - blasts of the tunica albuginea o v a r i i ( f i g . 20). Vesicles of comparable size were not as frequently observed i n c o n t r o l material. There i s not always a d i r e c t c o r r e l a t i o n between the size of a v e s i c l e and the number of f e r r i t i n molecules within i t . Often a v e s i c l e of large dimensions contains fewer molecules than a much smaller v e s i c l e . On average, 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 seem to be larger than other c e l l v e s i c l e s . This could indicate that they arise from fusion of smaller v e s i c l e s , as large v e s i c l e s are not seen to open to the c e l l a p i c a l surface d i r e c t l y . In contrast to the HRP experiments, f e r r i t i n i s not found i n v e s i c l e s or invaginations associated with the membranes bounding the i n t e r c e l l u l a r c l e f t s . This would seem to support the argument that v e s i c l e s from the a p i c a l surface probably do not empty into the c l e f t s . Even though large v e s i c l e s were not often seen to empty into the basement membrane, the appearance of f e r r i t i n i n the deeper regions of the ovary and 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 that vesicular transport 33 i s the method by which f e r r i t i n leaves the germinal e p i t h e l i a l c e l l s . The d i s t r i b u t i o n of free 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 f l u i d seems to be random. No preferred routes of tracer movement are evident. 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 less abundant than HRP and, because of i t s sparseness, does not assume an obvious gradient of d i s t r i b u t i o n l i k e that of HRP. Even though much more f e r r i t i n i s contained i n t r a c e l l u l a r l y within v e s i c l e s , no c e l l u l a r gradient of tracer d i s t r i b u t i o n i s evident. This i s due to 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 content both within a c e l l and from c e l l to c e l l . However, the depth to which f e r r i t i n penetrates the ovary increases as time of exposure to f e r r i t i n increases. This i s indicated by i t s presence within f i b r o b l a s t v e s i c l e s of successively deeper regions of the tunica albuginea ( f i g . 20) and within the f o l l i c u l a r f l u i d of developing f o l l i c l e s of the outer cortex ( f i g . 23). F e r r i t i n penetration thus seems to be a time-dependent process. 34 C . Figures Figure 1« Overview of germinal epithelium and part of under- l y i n g tunica albuginea o v a r i i . One germinal e p i - t h e l i a l c e l l i s indicated (GE). Note the i r r e g u l a r nuclear contour i n these c e l l s , the complex i n t e r - c e l l u l a r c l e f t s (arrows), and the underlying base- ment membrane of variable thickness. The presence of a c a p i l l a r y (C) very near to the surface i s also seen. Scale bar i n this and a l l succeeding micro- graphs indicates 1 /xm. Uranyl acetate and lead c i t r a t e staining. X 5.800. Figure 2J Scanning electron micrograph of the ovarian e p i t h e l i a l surface showing the regional d i s t r i - bution of c e l l shape. Sites of apparent i n d i v i d u a l c e l l necrosis are indicated (arrows). I t i s not known whether such s i t e s occur normally i n the germinal epithelium or whether they are ar t e f a c t u a l . X 420.  Figure 3» Typical cuboidal germinal e p i t h e l i a l c e l l s . C e l l on l e f t , which appears to be detached from r e s t of epithelium due to plane of sectioning, contains l i p i d inclusions ( a s t e r i s k s ) . Rough endoplasmic reticulum, ribosomes, and mitochondria are abundant. M i c r o v i l l i are also numerous. Note random arrange- ment of collagen f i b e r s i n basement membrane (BM) and highly variable course and appearance of i n t e r - c e l l u l a r c l e f t s . Uranyl acetate and lead c i t r a t e staining. X 10,600. Figure 4i Typical squamous germinal e p i t h e l i a l c e l l containing same organelles as cuboidal c e l l i n f i g . 3. A nucleolus (N) i s also present. Uranyl acetate and lead c i t r a t e staining. X 10,600. Inset shows Golgi 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 of variable content and electron density. A multivesicular body (arrow) i s also seen. Uranyl acetate and lead c i t r a t e s t a i n i n g . X 19,000.  Figure 5» Germinal epithelium. I n t e r c e l l u l a r c l e f t , showing complexity of i n t e r d i g i t a t i o n of adjacent c e l l sur- faces. Uranyl acetate and lead c i t r a t e staining. X 37.800. Figure 6« Germinal epithelium. Small punctate junctions, which may be gap junctions or macular t i g h t junctions, are indicated (arrows). Uranyl acetate and lead c i t r a t e staining. X 56,400. Figure 7* Germinal epithelium. Extended regions of c e l l approximation are shown (arrows). These may be gap junctions. Uranyl acetate and lead c i t r a t e s t a i n i n g . X 32,400. Figure 8i Germinal epithelium. Long region of apparent membrane fusion, creating appearance of a penta- laminar junction. Uranyl acetate and lead c i t r a t e s t a i n i n g . X 65,200. Figure 9» Germinal epithelium. Detailed structure of a junc- t i o n which appeared to be pentalaminar at a lower magnification. A d e f i n i t e space can be seen to separate the two external l e a f l e t s of apposing c e l l membranes. Uranyl acetate and lead c i t r a t e s t a i n i n g . X 235.000. 40 F i g u r e 10 i Scanning e l e c t r o n micrograph of the germinal e p i t h e l i u m , showing 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 . X 3,000. F i g u r e 11 i Scanning e l e c t r o n micrograph showing p i n o c y t o t i c i n v a g i n a t i o n s of a germinal e p i t h e l i a l c e l l a p i c a l s u r f a c e . M i c r o v i l l i can a l s o be seen. X 15,000.  Figure 121 Overall d i s t r i b u t i o n of HRP. Note i t s presence on ap 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 pino- c y t o t i c invaginations and vesic l e s of germinal e p i t h e l i a l c e l l s (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 of tunica albuginea o v a r i i . Uranyl acetate and lead c i t r a t e s t a i n i n g . X 5,800. Figure IJt E x t r a c e l l u l a r gradient of HRP. Density of HRP decreases from germinal e p i t h e l i a l c e l l (GE) a p i c a l surface through i n t e r c e l l u l a r e l e f t s , basement membrane, and in t e r s t i t i u m . Uranyl acetate and lead c i t r a t e staining. X 16,600. Inset shows i n t e r c e l l u l a r c l e f t s (arrows) of a cont r o l specimen, s i m i l a r l y stained with uranyl acetate and lead c i t r a t e , f or purposes of comparison re. electron 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 of 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 buffer washing. Note that HRP i s s t i l l found i n deeper regions of germinal epithelium and subjacent tissues. Uranyl acetate and lead c i t r a t e staining. X 14,500. Figure 15» Germinal epithelium. Note extended invagination of i n t e r c e l l u l a r c l e f t ( a s t e r i s k ) . Sectioning i n another plane could r e s u l t i n creation of an apparent HRP-containing v e s i c l e deep within c e l l . Uranyl acetate and lead c i t r a t e staining. X 24,300. Inset shows large "vesicle*" (arrow) near i n t e r - c e l l u l a r c l e f t to which are connected smaller v e s i c l e s Uranyl acetate and lead c i t r a t e s t a i ning. X 19,400. Figure 16» I n t e r c e l l u l a r c l e f t showing continuity of HRP from peritoneal surface above to basement membrane below. Tracer-containing v e s i c u l a r p r o f i l e s (arrows) may act u a l l y be pinocytotic invaginations s i m i l a r to those seen at the a p i c a l and basal surfaces of the germinal e p i t h e l i a l c e l l s . Uranyl acetate and lead c i t r a t e staining. X 73,200.  Figure 17 i Germinal epithelium. I n t e r c e l l u l a r c l e f t showing possible region of membrane fusion (arrow) near apical end. Note the deposition of HRP i n adjacent areas. Uranyl acetate and lead c i t r a t e staining. X 73,200. Figure 18i I n t e r c e l l u l a r c l e f t showing region of exclusion of HRP (arrow), suggesting that some junctions are not permeable to this tracer. Vesicle (V) i n lower r i g h t corner i s a v a r i a t i o n of that seen i n f i g . 15 inset. Uranyl acetate and lead c i t r a t e s t a i ning. X 73,200. 48 Figure 1 9 i Germinal e p i t h e l i a l c e l l (above) showing ve s i c l e s and multivesicular bodies containing f e r r i t i n (arrows). Free f e r r i t i n p a r t i c l e s are also seen i n the underlying basement membrane. Uranyl acetate staining. X 2 4 , 3 0 0 . Figure 2 0 i Overview of germinal epithelium (GE) and subjacent tis s u e s . The presence of large 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 (arrows) i n f i b r o b l a s t s (F) of the tunica albuginea o v a r i i i s well demonstrated. Uranyl acetate staining. X 1 9 , 4 0 0 .  Figure 21i F e r r i t i n i s present i n c a p i l l a r y endothelial c e l l (E),vesicles (arrows), and i n luminal plasma ( a s t e r i s k ) . Note p l a t e l e t (P) within lumen of c a p i l l a r y . Uranyl acetate staining. X 3 0 , 5 0 0 . Figure 22J A p i c a l surface of germinal e p i t h e l i a l c e l l . A possible sequence of the pinocytotic events underlying f e r r i t i n uptake i s shown (arrows). Uranyl acetate and lead c i t r a t e staining. X 88,400.  Figure 23* Overview showing germinal epithelium (GE), tunica albuginea o v a r i i , and granulosa c e l l s surrounding f o l l i c u l a r cavity (FC). Uranyl acetate and lead c i t r a t e s t a ining. X 4 , 7 0 0 . Inset shows f e r r i t i n i n f o l l i c u l a r f l u i d at ^ high magnification. Uranyl acetate and lead c i t r a t e s t a ining. X 117,000.  DISCUSSION 55 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. The increase i n surface area, i f the ovary i s to remain covered by the germinal epithelium, requires a change i n e p i t h e l i a l c e l l shape. The nature of thi s change i s an increase i n the area covered by a c e l l , i . e . , a c e l l must "stretch"' from a cuboidal to a squamous shape. I t i s possible that the convoluted infoldings of the i n t e r c e l l u l a r c l e f t s between cuboidal c e l l s may contribute to the required increase i n c e l l surface area. This i s d i f f i c u l t to assess, as the c l e f t s between squamous c e l l s are s t i l l quite convoluted. They do not appear to have become more d i r e c t i n t h e i r course, as might be expected. Some authors suggest that perhaps an increase i n e p i t h e l i a l c e l l number i s also involved i n e p i t h e l i a l accommodation to ovarian expansion (Wischnitzer, 1965? Weakley, 1969). However, no mitotic stages other than interphase were observed i n the germinal epithelium of this study, even at the apex of Graafian f o l l i c l e s , where such d i v i s i o n might be most expected. I t thus seems un l i k e l y that 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 could be a major response to an increase i n ovarian surface area. This i s not to imply that 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 c a p a b i l i t y to r e p l i c a t e by mitotic d i v i s i o n , merely that t h i s would not seem to be a normal response to ovarian volume increase. That e p i t h e l i a l c e l l s are s t i l l able to undergo mitosis i s indicated by the presence of centrioles i n association with the c i l i a seen i n the present study. Subsequent to ovulation the e p i t h e l i a l c e l l s must presumably undergo mitosis i n order to re- e s t a b l i s h the i n t e g r i t y of 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 should have been of quite common occurrence. To the contrary, no such regions were observed i n the specimens examined. As well as a passive a l t e r a t i o n i n e p i t h e l i a l c e l l shape, recent studies have implicated the possible involvement of an active component (Jeppesen, 1975)• Such a hypothesis i s supported by the observation i n the present study of microfilaments i n some germinal e p i t h e l i a l c e l l s , which suggests that the c e l l s may have an i n t r i n s i c a b i l i t y to contract. Post-ovulatory re-establishment of cuboidal c e l l shape could thus be explained on the basis of a c o n t r a c t i l e event. As a c o r o l l a r y to t h i s , c e l l "stretching" could be a function of the r e l a x a t i o n of 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 f e t a l guinea pig ovary, where i t has been proposed that they contract the s u p e r f i c i a l layer of the s t r a t i f i e d epithelium (Jeppesen, 1975). The r e s u l t of t h i s contraction i s that the more ba s a l l y situated c e l l s are forced into the gonadal anlage where they become associated with the sex cords as p r e f o l l i c u l a r c e l l s . Jeppesen noted that microfilaments are observed only when changes i n c e l l shape are occurring during development. This could explain why microfilaments are not often seen i n adult germinal e p i t h e l i a l c e l l s , as the epithelium has e s s e n t i a l l y completed i t s development. B. I n t e r c e l l u l a r Junctions Junctional 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 epithelium have been noted i n previous studies of both germinal epithelium and mesothelium. Focal regions of apparent membrane fusion observed i n t h i s study (see Results) appear s i m i l a r to junctions which have been i d e n t i f i e d i n the germinal epithelium of other species as t i g h t junctions . or junctional complexes (Papadaki and Beilby, 1 9 7 1 ) . " t y p i c a l junctional complexes (terminal bars)" (Gondos, 1 9 6 9 ) . or terminal bars (Wischnitzer, 1 9 6 5 ) . These terms are usually reserved to describe junctions which are, or have as one of t h e i r components, circumcellular zones of molecular occlusion. Weakley ( 1 9 6 9 ) was e x p l i c i t i n avoiding the term " t i g h t junction" when describing the junctions of hamster germinal epithelium. As well as fused f o c a l junctions, she also noted f o c a l regions where membranes did not seem to be fused but were separated by a small 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 of the f o c a l junctions of the present study. I t i s d i f f i c u l t to ? assess these junctions i n s t r u c t u r a l , and hence functional, terms with regard to the degree of membrane separation without res o r t i n g to more s p e c i a l i z e d techniques. For instance, freeze fracture studies of the f o c a l junctions which seem to be separated by a small i n t e r c e l l u l a r space could indicate whether or not these junctions are gap junctions. The appearance of membrane fusion could 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 sectioning through a junction, compounded by the torttpsity of the i n t e r c e l l u l a r c l e f t s . Such an i n t e r p r e t a t i o n could indeed explain some of the f o c a l fusion of adjacent germinal e p i t h e l i a l c e l l membranes seen i n this study. However, 59 examination of s e r i a l sections also suggests that some of these f o c a l points of fusion are i n fact quite r e a l and not a r t e f a c t u a l . Such fusion i s not extensive and i t i s doubtful that i t i s continuous about the entire periphery of a c e l l as an occluding junction. As well, i f some germinal e p i t h e l i a l c e l l s are joined by zonular t i g h t junctions, then one would expect to observe such junctions 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 epithelium i s to constitute an e f f e c t i v e b a r r i e r to d i f f u s i o n . In f a c t , i n t e r c e l l u l a r c l e f t s devoid of any junctional s p e c i a l i z a t i o n are sometimes noted. In l i g h t of the r e s u l t s of the present study with regard to tracer movement, i t i s extremely unlike l y that the areas of e p i t h e l i a l c e l l contact are t i g h t junctions i n the sense that they prevent i n t e r c e l l u l a r molecular d i f f u s i o n . The interpreta- t i o n given to the f o c a l fusion of membranes seen i n t h i s study i s thus the same as that given to junctions of s i m i l a r appearance i n mesothelium (Cotran and Karnovsky, 1968) and c a p i l l a r y endo- thelium (Karnovsky, 196?). Namely, these small points of membrane fusion are most l i k e l y maculae occludentes. or "spots of occlusion." Their function would thus seem to primarily involve c e l l adherence rather than regulation of molecular movement through the i n t e r c e l l u l a r spaces of the germinal e p i - thelium. T e l e o l o g i c a l l y , one might reason that the germinal epithelium does not constitute an impervious b a r r i e r which protects the ovary and i t s contained oocytes because the same junctions on which the effectiveness of such a b a r r i e r depends could also 60 make ovulation a more d i f f i c u l t process. That i s , such junctions could constitute a mechanical hindrance to the release of oocytes. Even i f such a b a r r i e r d i d allow ovulation, every time an oocyte was released p o t e n t i a l l y harmful substances could enter the ovary. These substances could diffuse from the peritoneal cavity through the e p i t h e l i a l break at the apex of the ruptured Graafian f o l l i c l e from which the oocyte was expelled. The substances would then be i n the cavity of the corpus luteum, and unless a l l the constituent c e l l s of that structure were joined by occluding junctions, which they do not seem to be, molecular movement through the ovary could continue unhindered. This would defeat the whole purpose of an impermeable epithelium, The s i t u a t i o n would be exacerbated i n the r a t , which i s poly- ovular, and would thus present numerous openings into the ovary from the peritoneal cavity through several ruptured Graafian f o l l i c l e s . C. M i c r o v i l l i The presence of m i c r o v i l l i has been noted i n previous studies of the germinal epithelium (Wischnitzer, 1965.' Gondos, 1969; Weakley, 1969? Papadaki and Beilby, 1971), as well as i n the present study. Studies of m i c r o v i l l i of the peritoneal mesothelium, which i s of the same embryological o r i g i n as the germinal epithelium, have also been conducted (Cotran and Karnovsky, 1968j Andrews and Porter, 1973). Most authors believe that m i c r o v i l l i serve to enhance the absorptive or secretory surface-to-volume r a t i o of 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 virtue of i t s numerous mi c r o v i l l i , may be involved i n the translocation of material either to or from the peritoneal cavity. Another interesting proposal for the function of peritoneal mi c r o v i l l i has been put forth by Andrews and Porter (1973)' They believe that the regions between adjacent mi c r o v i l l i entrap the slippery mucins secreted by the peritoneal meso- th e l i a l c e l l s . 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 result of such entanglement and binding would be a slippery liquid cushion layer which could function in protecting the underlying thin mesothelium from surface abrasion arising from normal movement of internal organs over one another." Such a mechanism could assist in the maintenance of the integrity of the germinal epithelium. Even though the epithelium may be protected in 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 mic r o v i l l i . This theory also allows the postulation of one possible function for the proteins presumably synthesized and secreted by the germinal epithelial c e l l s . These proteins could be a component of the serous exudate which lubricates the surfaces of the intraperitoneal viscera. Although no specific attempts were made to preserve or stain such a mucinous surface coating in the present study, there did from time to time appear to be amorphous remnants 62 of such a coating adherent to the peritoneal surface of the germinal epithelium. D. Protein Synthesis The germinal e p i t h e l i a l c e l l s are r i c h i n rough endoplasmic reticulum, mitochondria, and free ribosomes. Golgi complexes are also present. These features are often associated with synthetic metabolic a c t i v i t y , some of which could be concerned with l i p i d metabolism. In addition, the abundance of ribosomes and rough endoplasmic reticulum indicates a high degree of peptide synthesis. Usually, when ribosomes occur s i n g l y within the cytoplasm rather than as polysomes or rough endoplasmic reticulum, the manufactured peptides are retained f o r endogenous use (Lentz, 1971). Rough endoplasmic reticulum i n a c e l l i s commonly concerned with the manufacture of protein f o r export (Lentz, 1971). These observations on both free ribosomes and those associated with the endoplasmic reticulum are together suggestive of a high 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 with the synthesis of protein for export. The abundance of mitochondria also attests to the high energy requirements of these c e l l s , presumably f o r synthetic or transport functions. In c e l l s whose prime function i s protein export the Golgi complex i s prominent as the s i t e of packaging and concentration of proteins synthesized i n the rough endo- plasmic reticulum. Proteins are subsequently transported and released i n the form of membrane bound v e s i c l e s . As previously noted (see Results), the morphology of the Golgi complex varies 63 considerably from one germinal e p i t h e l i a l c e l l to another. Numerous smal 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 density and content are often seen i n the v i c i n i t y of the Golgi complex. Because such v e s i c l e s are a lso seen to be i r r e g u l a r l y scat tered through- out much of the r e s t of the cytoplasm, i t i s d i f f i c u l t to assess whether or not they are also a product of the a c t i v i t y of the Golgi complex. Weakley (1969) observed the indentat ion of the basal surface of the germinal 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 conta ining many ribosomes. There seemed to be no in tervening basement membrane and the c e l l s were apparently i n d i r e c t contact . She suggested that t h i s could indica te 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 c e l l s , or p r o t e i n t ransfer i n the opposite d i r e c t i o n . No such c y t o - plasmic processes were observed i n the present study. Considering the amount of peptide synthetic machinery i n the germinal e p i - t h e l i a l c e l l s , i t seems u n l i k e l y that these c e l l s would t r a n s f e r free amino acids ra ther than completed peptides . Also i n view of t h i s e p i t h e l i a l preponderance of synthet ic organelles and t h e i r presumed production of a large number of peptides , i t seems u n l i k e l y that f o l l i c u l a r c e l l s would be t ransport ing pep- t i d e s to the germinal e p i t h e l i a l c e l l s . I t would seem to be much more reasonable to suspect p r o t e i n t r a n s f e r to occur from the germinal e p i t h e l i a l c e l l s to e i ther the underlying 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 . The p o l a r i t y of i n t r a c e l l u l a r organelles i s not consistent enough to indica te any major route of secretory a c t i v i t y . 64 Autoradiographic studies may prove useful i n further i n v e s t i g a t i o n of protein synthesis, transport, and function. E. Lipids L i p i d inclusions s i m i l a r to those seen i n t h i s study have also been observed i n the germinal e p i t h e l i a l c e l l s of the hamster (Weakley, 1 9 6 9 ) and human (Papadaki and Beilby, 1 9 7 1 ) . I t i s possible that such inclusions are associated with s t e r o i d a l a c t i v i t y since t h e i r morphology i s s i m i l a r to that observed i n subjacent c o r t i c a l c e l l s . Germinal e p i t h e l i a l c e l l s are believed to give r i s e to f o l l i c u l a r c e l l s during development. A high degree of s t e r o i d a l a c t i v i t y i s manifest i n these f o l l i c u l a r c e l l s . Similar a c t i v i t y i n the germinal epithelium, although not as pronounced, could represent the retention of 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, 1969? Papadaki and Beilby, 1 9 7 1 ) . Certain enzymes have been histochemically l o c a l i z e d within germinal e p i t h e l i a l c e l l s . The concentration of 173-hydroxy- s t e r o i d dehydrogenase i s quite marked. Also present are the 16-a and 166 forms, the le v e l s of which are much lower ( B a i l l i e et a l . , 1 9 6 6 ) . These enzymes are c a t a l y t i c f o r s p e c i f i c reactions i n the metabolism of 1 6 - and 17-hydroxysteroids, which are probably of an estrogenic nature ( B a i l l i e et a l . , 1 9 6 6 ). I t i s d i f f i c u l t to reconcile the presence of these enzymes within the germinal e p i t h e l i a l c e l l s , as the c e l l s have not been found to demonstrate 3-hydroxysteroid dehdrogenase a c t i v i t y ( B a i l l i e et a l . , 1 9 6 6 ). This i s an es s e n t i a l enzyme i n the 6 5 i n i t i a l stages of metabolism of the'16- and 17-hydroxysteroids. I t would seem to be highly unlikely that the enzymes of l a t e r stages of the metabolic pathways would be present i n the absence of the enzyme required to synthesize t h e i r precursors. I t may be that the histochemical techniques used to investigate 8-hydroxysteroid dehydrogenase a c t i v i t y are not s e n s i t i v e enough to reveal i t s presence i n the germinal e p i t h e l i a l c e l l s . The p o s s i b i l i t y also exists of an alternate metabolic pathway which does not require t h i s enzyme. And f i n a l l y , the substrates f o r 173-» 16a-, and l6g-hydroxysteroid dehydrogenase c a t a l y s i s could be synthesized i n other c e l l s and transferred to the germinal e p i t h e l i a l c e l l s f o r f i n a l processing. Weakley (1969) noted that i n the germinal epithelium of the hamster there i s an increase i n the number of l i p i d inclusions with development. Such inclusions are present i n many embryonic tissues but usually disappear during d i f f e r e n t i a t i o n . Because of the increase i n l i p i d inclusions i n the e p i t h e l i a l c e l l s , she suggested that "they represent a s p e c i f i c product of the d i f f e r e n t i a t e d c e l l rather than nutrient material to be used by the developing tissue." The presence of l i p i d inclusions, t h e i r increasing numbers during development, and the l o c a l i z a - t i o n of s p e c i f i c enzymes within the germinal e p i t h e l i a l c e l l s a l l point 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, this involvement i s presumably of a much les s e r degree than i s seen i n f o l l i c u l a r and l u t e a l c e l l s . This i s attested to by the lower cytoplasmic l i p i d 66 content of e p i t h e l i a l c e l l s and by eit h e r the absence or very small number of u l t r a s t r u c t u r a l features usually associated with c e l l s whose prime function i s steroidogenic. These l a t t e r features include tubular mitochondrial c r i s t a e and smooth endoplasmic reticulum. I t i s possible that some smooth endo- plasmic reticulum may have been mistaken for small i n t r a c e l l u l a r v e s i c l e s . F. HRP Movement and L o c a l i z a t i o n The r e s u l t s of the present study indicate that the germinal epithelium of the r a t i s r e a d i l y permeable to HRP. The move- ment of HRP seems to be predominantly through the i n t e r c e l l u l a r c l e f t s . Vesicular transport of HRP across the germinal e p i - thelium could not be proven, even though there does seem to be a small but d e f i n i t e population of t r a c e r - f i l l e d cytoplasmic ve s i c l e s which do not appear to be connected to any of the c e l l surfaces. However, i n terms of the numbers of these v e s i c l e s , t h e i r s i z e , and t h e i r HRP content, i t would seem that the r o l e of ve s i c u l a r transport i n moving HRP and other molecules of a s i m i l a r s i z e across the germinal epithelium i s minor i n comparison to i n t e r c e l l u l a r d i f f u s i o n . These results are s i m i l a r to those found by Cotran and Karnovsky (1968) i n t h e i r study of rat mesothelium, which i s of s i m i l a r embryological o r i g i n to the germinal epithelium. The movement of HRP through the i n t e r c e l l u l a r c l e f t s i s probably i n the nature of a passive d i f f u s i o n process. Support i s lent to t h i s conclusion by the work of Cotran and 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 , quite s i m i l a r to the germinal epithelium. In mesothelium exposed to HRP ei t h e r during or af t e r f i x a t i o n there was s t i l l 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 HRP. Any active 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 obviously have been rendered non-functional, thereby leaving simple d i f f u s i o n as the only means by which HRP could be translocated. 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 to the ap i c a l surface or i n t e r c e l l u l a r c l e f t s or were i n close apposition to them, again suggestive of f i l l i n g by d i f f u s i o n (see Results). There were no tracer-containing v e s i c l e s deep within the mesothelial c e l l s . In addition, studies of f o l l i c u l a r permea- b i l i t y to in t r a v a s c u l a r l y - i n j e c t e d HRP have shown that 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 of f a t germinal epithelium a f t e r leaving the c i r c u l a t i o n (Anderson, W., 1972a). Thus there i s movement of HRP not only from the basement membrane toward the peritoneal surface but also i n the opposite d i r e c - t i o n , as revealed i n the present study. To explain these move- ments on the basis of the operation of active transport mechan- isms, one. would have to postulate the existence of either an active transport mechanism moving the same type of molecule b i d i r e c t i o n a l l y . 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 transporting the same type of molecule, but i n d i f f e r e n t directions, could be postulated. In eithe r case t h i s i s not l i k e l y , as i t would be of no obvious benefit to a c e l l or tissue. The r e s u l t s do suggest that 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 di r e c t i o n , and would thus seem to support a d i f f u s i o n theory f o r HRP movement through the germinal epithelium. 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 tracer by v e s i c l e s from the apical surface emptying t h e i r contents into the c l e f t s has already been noted,(see Results). This f a c t o r was also taken into account by Cotran and Karnovsky (1968). They showed that exposure of mesothelium to HRP eithe r during or subsequent to i n i t i a l f i x a t i o n (and preceding post-fixation).which would h a l t v e s i c u l a r transport, did not measurably reduce the amount of HRP i n the i n t e r c e l l u l a r c l e f t s . These re s u l t s demonstrate that i f there i s a vesicula r contribution to c l e f t f i l l i n g i t i s inconsequential. Presumably the same may hold true f o r the germinal epithelium. G. F e r r i t i n Movement and Lo c a l i z a t i o n The r e s u l t s suggest that the movement of f e r r i t i n through the germinal epithelium i s an i n t r a c e l l u l a r process mediated by vesicu l a r transport. A s i m i l a r cytoplasmic v e s i c u l a r trans- port of f e r r i t i n i s the sole means of f e r r i t i n movement through c a p i l l a r y and endothelial c e l l s subsequent to intravascular f e r r i t i n i n j e c t i o n s (Anderson, W., 1972a; Payer, 1975)- There i s no evidence whatsoever of i n t e r c e l l u l a r passage of f e r r i t i n through the germinal epithelium. It has been noted that free f e r r i t i n p a r t i c l e s are occa- s i o n a l l y seen within 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 . Their appearance i n 69 these locations i s regarded as artefactual f o r a number of reasons. F i r s t l y , these situations were observed infrequently i n t h i s study. I f the cytoplasm or the i n t e r c e l l u l a r c l e f t s were common s i t e s of f e r r i t i n movement, one would expect to observe free intracytoplasmic and i n t e r c e l l u l a r f e r r i t i n p a r t i c l e s on a constant basis. Such i s not the case. In addition, free f e r r i t i n p a r t i c l e s , when present within a c e l l , are not l i m i t e d i n occurrence to the cytoplasmic matrix. They may appear indiscriminately both within the matrix and within most c e l l organelles and i n or on the membranes of those organelles. This suggests that f e r r i t i n p a r t i c l e s may have been "smeared" across a section and are s u p e r f i c i a l l y situated. The source of these p a r t i c l e s cannot be d e f i n i t e l y established. They could be derived from sectioned v e s i c l e s which contain large numbers of f e r r i t i n molecules. Some of these molecules may have been displaced during the sectioning process to sub- sequently appear randomly over a section. Excessive f e r r i t i n at the peritoneal surface may also have been displaced by sectioning. F i n a l l y , the density of i n t r a v e s i c u l a r f e r r i t i n i s always sub- s t a n t i a l l y greater than the density of free f e r r i t i n , i n d i c a t i n g the overwhelming predominance of vesicula r transport. The penetration of f e r r i t i n through the epithelium and sub- jacent regions i s much slower than the movement of HRP. This i s l i k e l y a function of t h e i r d i f f e r i n g molecular size and weight and consequent differences i n t h e i r modes of movement. Such temporal differences have been noted i n other studies employing these two tracers (Payer, 1975). 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 of some developing f o l l i c l e s indicates that the f o l l i c u l a r basement membrane does not constitute a b a r r i e r to f e r r i t i n movement. This contradicts the r e s u l t s of a previous study (Anderson, W., 1972a). Using the same s t r a i n of r a t as was used i n thi s study, Anderson found that 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 to accumulate at the l e v e l of the f o l l i c u l a r basement membrane and did not pass through i t . No explanation can be put forth at present to r a t i o n a l i z e the observed differences i n f o l l i c u l a r permeability. The differences do not seem to be r e l a t e d to the time of exposure to f e r r i t i n , as the times i n the present study and i n Anderson's study were s i m i l a r . Anderson (W., 1972a), i n his study of f o l l i c u l a r permeability, used a vari e t y of intravascular tracers. He shows the presence of Thorotrast (molecular diameter 70 A) i n i n t r a c e l l u l a r c l e f t s and large phagocytic vacuoles of the germinal epithelium. These vacuoles are i n close proximity to the c l e f t s and some appear as though they could be sections through i r r e g u l a r i t i e s i n these c l e f t s (see Results). It i s suggested by Anderson that Thorotrast does not pass through the entire length of the c l e f t s into the peritoneal cavity, due to the presence of junctions i n the c l e f t s . Nevertheless, f o c a l and extended junctions were often observed at the basal end of i n t e r c e l l u l a r c l e f t s i n material examined i n the present study. Presumably these types of junctions would also have been present i n the experimental animals used by Anderson, as some of his animals were of the same s t r a i n as used herein. I f Thorotrast passed 71 either through or around these basally-situated junctions, then i t would be l i k e l y that i t would also pass through junctions at the peritoneal ends of the c l e f t s , as there are no zonal regions of occlusion there. Additionally, as previously mentioned, some c l e f t s appear to have no junctional s p e c i a l i - ; zations at a l l . I t would thus be expected that i f Thorotrast entered the basal end of such a c l e f t , i t would eventually diffuse to the peritoneal end and into the peritoneal cavity. The upper l i m i t of molecular size i n terms of passive i n t e r - o c e l l u l a r d i f f u s i o n would therefore seem to be between 70 A (Thorotrast) and 110 A ( f e r r i t i n ) . The 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 current study p a r a l l e l s i n many respects that seen i n previous studies of the ovary i n which f e r r i t i n was introduced v i a inatravascular i n j e c t i o n (Anderson, W., 1972a; Payer, 1975). The observation that there are more large 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 control material suggests that f e r r i t i n may have an inducing e f f e c t on v e s i c u l a r formation. 72 CONCLUSION On the basis of i t s u l t r a s t r u e t u r a l features, the germinal epithelium appears to be a highly active tissue. It possesses structures suggestive of an absorptive and/or secretory capa- c i t y ( m i c r o v i l l i ) , a marked protein synthetic a c t i v i t y ( r i b o - somes and rough endoplasmic reticulum), and possible 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 epithelium may be a c t i v e l y or passively involved i n the movement of tracers has also been demonstrated. Exposure of the peritoneal surface of the ovary to HRP and f e r r i t i n , and subsequent electron microscopic l o c a l i z a t i o n of these tracers, indicate that there are two d i s t i n c t routes of molecular movement from the peritoneal cavity across the germinal epithelium, according to the size of the molecule translocated. o Molecules of the order of 40 A diameter, such as HRP, appear to f r e e l y diffuse through the i n t e r c e l l u l a r c l e f t s of the epithelium. o Larger molecules, comparable i n size 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 across the germinal epithelium. "Such a cytoplasmic route could provide a means of screening substances destined for the underlying tissues, or of a l t e r i n g them chemically before they are allowed to proceed further (Weakley, 1969)." Because only the f e r r i c hydroxide core of f e r r i t i n i s seen with the electron microscope, i t i s not possible to observe whether changes i n the apoprotein portion of the f e r r i t i n molecule occur during i t s passage through the epithelium. Such a study would probably be amenable to investigation by immunohistochemical techniques. 73 I t can be concluded that there i s ready access of substances the size of f e r r i t i n or smaller into the substance of the ovary through the germinal epithelium. From th i s i t follows that substances which permeate the epithelium and subjacent tissues may, i f not al t e r e d during t h e i r t r a n s e p i t h e l i a l passage, influence the normal development of the oocyte. The effects exerted on the oocyte by these substances may or may not be detrimental. At the l e v e l of the epithelium, smaller 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 subject to any biochemical screening procedure which could either prevent t h e i r passage or a l t e r t h e i r molecular structure. Hence t h e i r e f f e c t s , i f any, on the oocyte would be d i r e c t . Larger molecules, which must pass through the e p i t h e l i a l c e l l s , could be subject to intracytoplasmic modification of t h e i r structure, or jtheir passage could be prevented e n t i r e l y . The magnitude of the e f f e c t of these molecules on the oocyte would be a function of the degree of 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 molecular structure. If an exogenous substance enters the peritoneal cavity, the possible effects of such a substance on the oocyte must be examined at several stages. F i r s t l y , i n terms of adverse e f f e c t s , foreign; molecules could interfere with normal oocyte development and metabolism. This could lead to a reduction i n oocytic v i a b i l i t y and a subsequent f a i l u r e of the oocyte , to reach maturity. Secondly, such molecules could a l t e r the oocytes, but i n a less r a d i c a l manner. Oocytic development, ovulation, and f e r t i l i z a t i o n could occur, but the zygote so 74 formed might not be viable, leading to an increased rate of spontaneous abortion. F i n a l l y , the effects of exogenous molecules could be so subtle as to allow development of the f e r t i l i z e d oocyte to term. However, the resultant progeny could manifest congenital abnormalities of varying degrees of s e v e r i t y due to alt e r a t i o n s i n the oocyte induced by the exogenous molecules. In terms of the possible c l i n i c a l s i g nificance of the findings of t h i s study, there are a number of situations, i n which exogenous molecules may enter the peritoneal cavity and thus come into contact with the germinal epithelium. Most of these si t u a t i o n s are associated with the entry of bacteria into the peritoneal cavity. The bacteria are the actual source of the exogenous molecules, which they release as either exotoxins or endotoxins. Positive 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 size found to be sim i l a r to enzymes i n general (Davis et a l . , 1973). The normal range of enzyme molecular weight i s from 12,000 to more than 1 m i l l i o n (Lehninger, 1970). I t would then appear that of the known b a c t e r i a l exotoxins, a number are of a molecular weight less than that of f e r r i t i n (4§2,000) . They may thus be able to pass through the germinal epithelium. Another source of exogenous molecules could be the digestive t r a c t subsequent to perforation of an organ. This would allow not only bacteria to enter the peritoneal cavity, but also various products of digestion and excretion. The routes by which toxin-producing bacteria and non-bacterially derived 75 exogenous substances may enter the peritoneal cavity are enumerated below ( E l l i s and Calne, 1976)» 1. from the exterior v i a an i n f e c t i o n at laparotomy or a penetrating wound. 2. from intra-abdominal viscera t a) gangrene of a viscus, 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 or i n f a r c t i o n of the intestine, b) perforation of a viscus, e.g. perforated duodenal ulcer, perforated appendicitis, rupture of intestine from trauma. c) post-operative leakage of an i n t e s t i n a l suture l i n e . 3. v i a the blood stream as part of a septicemia (pneumo- coccal, streptococcal, or staphylococcal). 4. 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 puerperal i n f e c t i o n . I t i s thus seen that the germinal epithelium i s a metabolically and s y n t h e t i c a l l y active tissue. Whether or not i t i s capable of modifying some of the molecules to which i t i s permeable remains to be demonstrated. Such processes could be of considerable significance i n the maintenance of a normal environment f o r oocytic development, especially as there are d e f i n i t e situations i n which exogenous molecules may enter the peritoneal cavity. 76 LITERATURE CITED Ainsworth, S.K. and M.J. Karnovsky. 1971. An ultrastruetural staining method for enhancing the size and electron opacity of f e r r i t i n in thin sections. J. Histochem. Cytochem. 20t 225-229. Anderson, E. 1967. Observations on the uptake of HRPO by the developing oocytes of the rabbit. J. Cell Biol. 35» 160 A. Anderson, E. 1972. The localization of acid phosphatase and the uptake of HRP in the oocyte and f o l l i c l e c e l l s of mammals. In« Oogenesis. J.D. Biggers and A.W. Schuetz, eds. University Park Press, Baltimore, pp. 87-117. Anderson, W.A. 1972a. 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