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Control of adrenocortical cytodifferentiation in vitro by metachromatic extracellular matrix Slavinski, Eva Ann 1976

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CONTROL OF ADRENOCORTICAL CYTODIFFERENTIATION IN VITRO BY METACHROMATIC EXTRACELLULAR MATRIX by EVA ANN SLAVINSKI B.Sc, The University of B r i t i s h Columbia, 1972. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the department of Zoology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1976. © Eva Ann Slavinski 1976 In p resent ing t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements fo r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e fo r reference and study. I f u r t h e r agree t h a t permiss ion for e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . Department of - t i o o V Q g ^ The U n i v e r s i t y of B r i t i s h Columbia 207 5 Wesbrook Place Vancouver, Canada V6T.1W5 ABSTRACT Methods of propagating homogeneous populations of f u n c t i o n a l adult r a t adrenal c o r t i c a l c e l l s i n monolayer cultures as either . d i f f e r e n t i a t e d e p i t h e l i a l - l i k e c e l l s or as p a r t i a l l y d i f f e r e n t i a t e d f i b r o b l a s t - l i k e c e l l s were developed. Further work has elucidated the l o c a l environmental parameters c o n t r o l l i n g adrenal c o r t i c a l function and has suggested the mechanism(s) by which these parameters might act. B r i e f l y , c e l l s that were subcultured by mechanical d i s s o c i a t i o n from confluent primary adrenal cultures and grown i n medium containing horse serum, assumed an e p i t h e l i a l - l i k e morphology and resembled adrenal c o r t i c a l parenchyma in vivo i n corticosterone production and s t e r o i d -ogenic, l i p o l y t i c ^response to adrenocorticotrophic hormone. C e l l s that were subcultured by t r y p t i c d i s s o c i a t i o n and grown i n medium containing f e t a l c a l f serum, assumed a f i b r o b l a s t - l i k e morphology and exhibited: c h a r a c t e r i s t i c s of both connective ti s s u e and adrenal c o r t -i c a l t i s s u e : . c e l l s produced metachromatic e x t r a c e l l u l a r matrix and c o l l -agen but funlike f i b r o b l a s t s of connective ti s s u e o r i g i n , they uniformly stained for. ;steroid and pentose shunt dehydrogenases as well as l i p i d , .•^produced small amounts of corticosterone and responded steroido-g e n i c a l l y and morphologically to adrenocorticotrophic hormone. The ste r o i d production of these f i b r o b l a s t - l i k e c e l l s was lower by 1-2 orders of magnitude than the st e r o i d production of e p i t h e l i a l - l i k e adrenal c e l l s , a degree of c y t o d i f f e r e n t i a t i o n that i s reminiscent of the p r o t o d i f f e r e n t -iated state that occurs during the morphogenesis of many embryonic t i s s u e s . Several observations suggested that c e l l morphology and the presence of metachromatic e x t r a c e l l u l a r matrix (MECM) were correlated«with' cyto-i i i d i f f e r e n t i a t i o n i n terms of steroid production. Addition of adreno-c o r t i c o t r o p i c hormone (ACTH) to f i b r o b l a s t - l i k e adrenal c e l l s over 3 days generally caused a disappearance of MECM and the development of an e p i t h e l i a l - l i k e morphology: the magnitude of steroidogenic response to ACTH correlated with the degree of MECM disappearance. Conversely, the addition of medium containing f e t a l c a l f serum (FCS-medium) to monolayers of e p i t h e l i a l - l i k e adrenal c e l l s caused modulation 3 to a f i b r o b l a s t - l i k e form and function within 24 h. A concomitant drop i n steroid production and a large increase i n MECM production occurred. In order to assess the influence of MECM on adrenal c o r t i c a l cytodifferentiation more d i r e c t l y , adrenal e p i t h e l i a l - l i k e c e l l s were exposed to (a) 6-diazo-5-oxo-L-norleucine (DON) , a drug which i n h i b i t s glycosaminoglycan synthesis, together with FCS-medium and (b) hyaluronic acid as an exogenous source of ex t r a c e l l u l a r matrix. E p i t h e l i a l - l i k e c e l l s exposed to DON i n FCS-medium did not produce MECM, did not develop a f i b r o b l a s t - l i k e morphology, did not migrate away from one another and continued to produce steroids at r e l a t i v e l y high levels. Cells exposed to hyaluronic acid (in medium containing horse serum) developed a f i b r o b l a s t - l i k e form and produced only minute amounts of steroid. The effect of this polyanion on adrenal c e l l morphology and function could be mimicked by growing c e l l s on surfaces which had been sulphonated to a negative charge density of approximately 170 charges per 100 $'2 . These observations implicated components of the ext r a c e l l u l a r matrix i n l i m i t i n g adrenal c o r t i c a l cytodifferentiation and suggested that th e i r influence was extra c e l l u l a r and en t i r e l y due to charge. The present study demonstrates that c e l l s exist within the adult rat adrenal cortex which are s t i l l capable of displaying responses to environmental parameters, such as ext r a c e l l u l a r matrix, that are i v c h a r a c t e r i s t i c of mesodermal embryonic t i s s u e s . Although the p h y s i o l -o g i c a l counterpart of these adrenal c o r t i c a l c e l l s w i t h i n the a d u l t cortex has not been d e f i n i t i v e l y i d e n t i f i e d , u l t r a s t r u c t u r a l and f u n c t i o n a l c h a r a c t e r i s t i c s suggest an o r i g i n from capsular t i s s u e , which has been i m p l i c a t e d as a stem c e l l source in vivo during a d r e n a l c o r t i c a l regen-e r a t i o n . MECM, which surrounds capsular c e l l s in vivo, may w e l l i n h i b i t t h e i r overt e x p r e s s i o n o f adult adrenal c o r t i c a l phenotype and thus provide a s t o r e of p o t e n t i a l adrenal c o r t i c a l parenchymal c e l l s capable of e x p r e s s i n g t h e i r a d u l t phenotype upon d i s s o l u t i o n of MECM, an event that could occur i n response to very high ACTH l e v e l s . ACKNOWLEDGEMENT S v I wish to express my sincere appreciation to Dr. Nelly Auersperg f o r her advice, guidance, tolerance and encouragement throughout t h i s i n v e s t i g a t i o n , and to Drs. A. Burton, C.V. Finnegan and A.M. Perks f o r t h e i r h e l p f u l suggestions during t h i s investigation.and for c r i t i c a l examination of the manuscript. I wish -to p a r t i c u l a r l y thank Dr. Burton for his c r i t i c a l advice during the preparation of manuscripts submitted for p u b l i c a t i o n and to Dr. R.L. Noble, Director of the Cancer Research Centre, for the opportunities and f a c i l i t i e s to carry out this research. I also wish to thank Ms. Denise McClellan for her help and advice. During the tenure of t h i s i n v e s t i g a t i o n , the author was the r e c i p -ient of a Univ e r s i t y of B r i t i s h Columbia Fellowship ..and an H.R. MacMillan Family Fellowship. This research was supported by grants from the National Cancer I n s t i t u t e to Dr..N. Auersperg. v i TABLE OF CONTENTS' P ag e ABSTRACT i i ACKNOWLEDGMENTS v TABLE OF CONTENTS v i LIST OF TABLES .C v i i i LIST OF FIGURES . x i INTRODUCTION . 1 MATERIALS AND METHODS 9 I. C e l l J c u l t u r e 9 II . Histochemistry 16 " I I I . S teroid determinations 18 IV. Experimental manipulation of secondary cultures . . . 26 RESULTS/.' 34 ' I v G r o w t h conditions 34 II . Functional properties 46 II I . Ultrastructure?,of adrenal c o r t i c a l c e l l s i n HS-medium and i n FCS-medium with and without ACTH :", : 68 IV. The ro l e of serum supplements, growth surfaces and subculture techniques i n determining culture morphol-ology, c e l l movement, MECM production and s t e r o i d secretion 91 DISCUSSION 126 I. O r i g i n and nature of secondary cultures of adrenal c o r t i c a l c e l l s 126 v i i page I I . The control of adrenal c o r t i c a l c e l l steroidog-enesis, morphology and movement i n v i t r o by ext r a c e l l u l a r matrix (MECM). . . 134 I I I . Conclusions . . . . 140 SUMMARY 142 LITERATURE CITED 145 APPENDIX I. Procedure for A5-3£-hydroxysteroid dehydrogenase . . . 156 I I . Chromatographic behavior of radiometabolites prod-uced by adrenal cultures and muscle fascia fibrob-la s t s 157 I I I . Chromatographic procedures 159 IV. Preparation of c e l l s for transmission electron microscopy 160 V. Calculations for determining charge density of of sulphonated p e t r i dishes 161 v i i i LIST OF TABLES Table page 1. The effect of different l o t s of horse serum on the a b i l i t y of secondary monolayer fragments, obtained from confluent primary culture, to attain an epith-e l i a l morphology 11 2. Influence of the addition of bovine serum albumin (fraction V) on fluorogenic steroid production of adrenal cultures 13 3. Simplified chart for mucin i d e n t i f i c a t i o n 17 4. Re c r y s t a l l i z a t i o n data of steroid metabolized by adrenal e p i t h e l i a l - l i k e and f i b r o b l a s t - l i k e c e l l s from [4- 1 1 +C]pregnenolone 20 5. The influence of the strain,age,sex of rat on (a) primary'cuLlturemmorphology. and growth and.(b) the a b i l i t y of explanted adrenal c e l l fragments to at t a i n an e p i t h e l i a l morphology when grown i n secondary culture i n HS-medium 37 6. Morphology of secondary adrenal cultures: influence of the serum supplement, method of c e l l diss-ociation and c e l l density of primary cultures at time of subculture 39 7. The influence of the extent of dissociation.on the a b i l i t y of secondary monolayers to attain an e p i t h e l i a l morphology . . . . . 42 8. Staining characteristics of secondary cultures adrenal f i b r o b l a s t - l i k e c e l l s maintained i n FCS-medium 51 9. Effect of ACTH on the conversion of [4- 1 ^ p r e g -nenolone to i d e n t i f i e d steroids by stationary c u l -tures of adrenal e p i t h e l i a l - l i k e , adrenal fibrob-l a s t - l i k e and muscle f a s c i a c e l l s 53 10. [4- 1 ^^pregnenolone metabolism, by "cloned" adrenal f i b r o b l a s t - l i k e c e l l s i n FCS-medium 57 11. Effect of ACTH on the secretion of endogenous fluorogenic steroid by adrenal e p i t h e l i a l - l i k e , f i b r o b l a s t - l i k e c e l l s and muscle f a s c i a fibroblasts . . 59 Table i x page 12. The e f f e c t of LH on s t e r o i d production, c e l l d i v i s i o n and MECM production of adrenal c o r t i c a l f i b r o b l a s t - l i k e c e l l s i n FCS-medium 61 13. The influence of ACTH on adrenal c o r t i c a l c e l l d i v i s i o n i n the presence of FCS-medium or HS-medium . . 64 14. The r e l a t i o n s h i p among secondary adrenal culture morphology, presence of MECM and endogenous s t e r o i d production of cellsimaintained i n FCS-medium i n the presence of ACTH 65 15. Outline of experiment, designed.to assess the cont-inued a b i l i t y of adrenal c o r t i c a l monolayers to., a l t e r morphology and f u n c t i o n a l expression when grown i n FCS-medium or HS-meddtum 92 16. The e f f e c t of FCS on the phenotypic expression of adrenal c o r t i c a l e p i t h e l i a l - l i k e c e l l s 96 17. A l t e r a t i o n of [4- 1 4C]pregnenolone metabolism a f t e r the addition of FCS-medium to adrenal e p i t h e l i a l -l i k e c e l l s 100 18. The e f f e c t of DON on the fluorogenie s t e r o i d production, c e l l morphology and MECM production of adrenal e p i t h e l i a l - l i k e c e l l s exposed to FCS-. medium 101 19. " The e f f e c t of serum, DON and modified substrata on adrenal c o r t i c a l c e l l movement 105 20. . The influence of added glycosaminoglycans on st e r o i d production, c e l l morphology, and MECM production of adrenal e p i t h e l i a l - l i k e c e l l s 110 21. Influence of pH of Toludidine blue solution, on the s p e c i f i c i t y of st a i n i n g sulphonated dishes I l l 22. The influence of treated growth surfaces on the st e r o i d production, MECM production and culture morphology of adrenal c o r t i c a l e p i t h e l i a l - l i k e c e l l s i n HS-medium . . 115 23. M i t o t i c index and MECM production of adrenal c o r t -i c a l c e l l s grown on sulphonated surfaces i n FCS-medium 117 X Table page 24. The effect of mechanical dissociation of mono-layer fragments obtained from confluent primary cultures on c e l l morphology, fluorogenic steroid production and. MECM production 123 25. Effect of colcemid on the [4-li+C]pregnenolone. metabolism of adrenal c o r t i c a l f i b r o b l a s t - l i k e c e l l s . . . 124 26. The influence.of colcemid on endogenous, steroid production of adrenal c o r t i c a l c e l l s 125 x i LIST OF FIGURES Figure page I. Conversion of c o l c h i c i n e to lumicolchicine i n the presence of u l t r a v i o l e t l i g h t . . . . 33 1. Primary cultures of normal adrenal corticalT.cells . . 35 2. Secondary cultures of adrenal c o r t i c a l c e l l s 40 3. Lifespan of e p i t h e l i a l - l i k e , f i b r o b l a s t - l i k e adrenal c e l l s and muscle f a s c i a f i b r o b l a s t s 44 4. Histochemistry of adrenal and muscle f a s c i a c e l l s . . 47 5. Adrenal mono-layers stained with t o l u i d i n e blue . . . . 49 6. Increase i n pregn-5-ene-3g!j*20a-diol with increasing c e l l number 56 7. Influence of ACTH on adrenal c e l l morphology 62 8. Influence of ACTH on adrenal c e l l morphology . . . . 62 9. U l t r a s t r u c t u r e of adrenal e p i t h e l i a l - l i k e c e l l s without ACTH 69 10. •; . U l t r a s t r u c t u r e of adrenal e p i t h e l i a l - l i k e , c e l l s treated with ACTH for 1 day 72 11. U l t r a s t r u c t u r e of adrenal e p i t h e l i a l - l i k e c e l l s treated with ACTH for 3 days • . . 77 12. U l t r a s t r u c t u r e of adrenal e p i t h e l i a l - l i k e c e l l s treated with ACTH for 3. and 5 days 73 79 13. Ul t r a s t r u c t u r e of adrenal f i b r o b l a s t - l i k e c e l l s without ACTH 83 14. U l t r a s t r u c t u r e of adrenal f i b r o b l a s t - l i k e c e l l s grown with ACTH for 1 day 86 15. Ul t r a s t r u c t u r e of adrenal f i b r o b l a s t - l i k e c e l l s grown with ACTH for 3 and 5 days 88 16. The morphology of adrenal c o r t i c a l c e l l s i n FCS-medium and i n HS-medium a f t e r up to 3 passages . . . . 93 17. E f f e c t s of FCS-medium on culture morphology of adrenal e p i t h e l i a l - l i k e c e l l s 97 x i i Figure page 18. The effect of DON added with FCS-medium on the culture morphology of adrenal e p i t h e l i a l - l i k e c e l l s . . 103 19. Adrenal e p i t h e l i a l - l i k e c e l l s exposed to DON and FCS-medium. Cultures stained with toluidine blue 106 20A Sulphonation of growth surfaces at 55°C and the effect on c e l l attachment 112 20B Effect of glutaraMdehyde f i x a t i o n on c e l l attach-ment on charged surfaces 113 21. Scanning electron microscopy of growth surfaces . . . 120 1 INTRODUCTION During the past several decades, b i o l o g i s t s have examined endogenous and to a lesser extent exogenous events con t r o l l i n g the overt expression of type-specific c h a r a c t e r i s t i c s , a phenomenon loosely described as c e l l d i f f e r e n t i a t i o n . However, as a result of inconsistent use of a precise d e f i n i t i o n , t h i s term has been applied to a variety of mechanistically unrelated phenomena (1-9). Therefore, prior to discussion of the environmental parameters that appear to regulate many of the events during development, an attempt w i l l be made to c l a r i f y the current usage of the term, c e l l d i f f e r e n t i a t i o n . Embryonic development may be viewed as the formation of pop-ulations of c e l l s of different phenotypes from a common genotype precursor. Tissue transplantation experiments (10,11) as w e l l as more recent in vitro experiments (12) suggest that early i n develop-ment many c e l l s have the capacity to form a variety of tissues. As development proceeds, however, this capacity i s increasingly l o s t so that at some point i n the developmental sequence, c e l l s appear to be programmed to exhibit one set of type-specific characteristics only. Such a process i s generally considered to be i r r e v e r s i b l e because c r i t i c a l evidence demonstrating loss of type-specific t r a i t s with the subsequent appearance of a set of characteristics s p e c i f i c for another c e l l type has not been reported. Tissue s p e c i f i c chara-c t e r i s t i c s are often overtly expressed long after such i r r e v e r s i b l e s p e c i f i c a t i o n has occurred (13). Further, i t appears that the prod-uction of these type-specific characteristics i s amplified i n d i s t -2 i n c t l y regulated phases (14) and, f i n a l l y , modulated i n the adult by a variety of environmental s t i m u l i (15). Each of these events has at one time or another been referred to as c e l l d i f f e r e n t i a t i o n even though there i s increasing evidence to suggest that each step involves a mechanistically different process (1,5,16-20). Holtzer (6) has suggested that the term c e l l d i f f e r e n t i a t i o n be applied to the process involving the emergence of daughter c e l l s that synthesize molecules the i r mother c e l l did not and that subsequent events alter i n g the extent of this production be regarded as physiol-ogical regulation of phenotypic expression. Accordingly, the events that lead to conversion of a hematocytoblast which does not prod-uce detectable hemoglobin, to an e r y t h r o b l a s t w h i c h does, i s provided as an example of c e l l d i f f e r e n t i a t i o n . The increased prod-uction of type-specific enzymes during pancreatic development des-cribed by Sanders and Rutter (17) i s referred to as an example of the physiological regulation of already differentiated cells.However, Weiss (16) points out that the events leading to the r e s t r i c t i o n of a c e l l genotype are l i k e l y ^ - t o l d i f f e r from the mechanism(s) allowing overt expression of type-specific characteristics. Therefore neither of the above examples adequately represents the process of c e l l d i f f e r e n t i a t i o n . Weiss has suggested that the term c e l l d i f f e r e n t i a t i o n be r e s t r i c t e d to describe the "uni-d i r e c t i o n a l and i r r e v e r s i b l e transformation which engenders true constitutional d i f f e r e n t i a l s or alterations i n type-specific chara-c t e r i s t i c s among the emerging daughter c e l l s " and that subsequent events involving translation of covert to overt type-specific chara-c t e r i s t i c s be defined as cytodifferentiation. 3 In an attempt to unify the current usage of c e l l d i f f e r e n t i a t i o n , the following d e f i n i t i o n s of the above development events w i l l be used i n t h i s report. C e l l d i f f e r e n t i a t i o n w i l l be used s t r i c t l y i n the context of Weiss' d e f i n i t i o n while cytodifferentiation w i l l be used to refer to the process temporally following c e l l d i f f -erentiation and culminating i n the production of type-specific characterics exhibited by mature c e l l s . This process w i l l be regarded as being composed of three discrete regulatory steps as proposed by Rutter (14). It has been known for some time that c e l l - c e l l interactions amongst homotypic and heterotypic populations of c e l l s are required for expression of type-specific characteristics during development. The nature and mechanism(s) of homotypic interactions are not well characterized although several investigators have suggested £hat. junction formation (21) or glycosylation of c e l l surfaces (22) i s involved. Heterotypic c e l l interactions can be , somewhate a r b i t r a r i l y , divided into l o c a l i z e d effects such as epithelial-mesenchymal interactions and effects exerted over a considerable distance such as the p i t u i t a r y control of a variety of organs. The l a t t e r i n t e r -actions encompass hormonal effects and appear to regulate late embryonic events (Rutter's t e r t i a r y regulatory step) and adult production of type-specific products. The mechanism of extra-c e l l u l a r to i n t r a c e l l u l a r information transfer by hormones i s f a i r l y well understood (5,23) Short range epithelial-mesenchymal interactions appear to be ubiquitous i n guiding c y t o d i f f e r e n t i a t i o n i n the embryo. As early as 1920, i t was realized that the i n i t i a t i o n 4 of nervous system formation i n the ectoderm depended upon the presence of the underlying mesoderm (11). Since then, the large number of organs i n which epithelial-mesenchymal i n t e r a c t i o n s have been demon-strated, suggests that such r e l a t i o n s h i p s are a general c h a r a c t e r i s t i c of c y t o d i f f e r e n t i a t i o n . The epit h e l i a l w h i c h have been shown to dep-end on underlying mesoderm for the expression of t y p e - s p e c i f i c c h a r a c t e r i s t i c s are derived from a l l three primary germ layers. Included are the skin ('2'4^ 5, epidermal d e r i v a t i v e s (25), mammary gland (26), s a l i v a r y gland (27), lens (28),.uretefiicc bud (29), thymus (30), p i t u i t a r y (31), thyroid (32), lung and pancreas (33). The mechanism(s) by which mesenchjfife passes morphogenetic information to a r e a c t i v e epithelium are net known. Suggestions as to the i d -en t i t y of the active factors have included d i r e c t c e l l - t o - c e l l surface i n t e r a c t i o n s (22,34,35), passage of RNA or another inform-ation-containing molecule that d i r e c t l y affects, c e l l u l a r metabolism (36) and passage of e x t r a c e l l u l a r matrix (34-38). Evidence f o r the passage of molecules that might enter the re a c t i v e epithelium and d i r e c t l y a f f e c t c e l l u l a r metabolism derives from radioactive studies, c e l l extracts and use of immunofluorescence (36). The i d e n t i t y of these molecules i s generally unkown as i s the r e l a t i o n -ship of such passage to induction of c y t o d i f f e r e n t i a t i o n . RNA has been suggested to pass between the i n t e r f a c i n g heterotypic tissues (25):. although Wessells has shown that l a b e l l e d RNA did not pass from mesenchyme to e p i t h e l i a l t i s s u e i n the pancreas (39). Nevertheless, granules' thought to represent or contain RNA, based on the i r s e n s i t i v i t y to RNAse,have.(been observed between ectoderm and mesoderm p r i o r to neuralation as^lwell as tooth development, and I'.t has been suggested that such RNA may contain the information necessary f o r the inductive 5 event to proceed (25). Grobstein's o r i g i n a l suggestion (40), that extracellular or interface material might be the active factor guiding e p i t h e l i a l c y t o d i f f e r e n t i a t i o n opened an entire new v i s t a of possible factors controlling c e l l and cyto-differentiation. In almost a l l e p i t h e l i a l -mesenchymal interactions, glycosaminoglycan and collagen deposition could be observed at the surface of the reacting e p i t h e l i a l tissue suggesting the production of such components either by the epith-elium or mesenchyme or both might be the ubiquitous guiding factor i n these interactions. Grobstein's work suggested the active factor during s a l i v a r y gland histogenesis to be collagen although t h i s has recently been questioned by Bernfield (37). Rather, i t appears that both collagen production by the mesenchyme and glycosaminoglycan synthesis by the epithelium are required for subsequent histogenesis (37,40). Since Grobstein's o r i g i n a l suggestion, the p o s s i b i l i t y that ex t r a c e l l u l a r matrix components guide events during embryo-genesis other than histogenesis has been investigated and interest i n determining and corelating the type of glycosaminoglycan with inductive events e a r l i e r i n development have increased (41,42). Thus collagen substratum has been shown to substitute for mesen-chymal tissue during corneal cytodifferentiation (8) and chondroitin sulphate has been shown to substitute for notochord i n promoting somite chondrogenesis (38). Other components of the extr a c e l l u l a r matrix have been implicated as possessing tissue s p e c i f i c , devel-opmentally in s t r u c t i v e properties i n a variety of other events during embryogenesis including c e l l migration patterns, mineralization, c a l c i f i c a t i o n , , myoblast fusion (42) and, i n the adult, wound healing repair and regeneration (25, 43,44). Hyaluronic acid,ain^paf-tii'cular, 6 appears to be ubiquitous i n i t s a b i l i t y to promote and control mesodermal c e l l migration and cytodifferentiation during both dev-elopment and regeneration (44 -48) . The mechanism(s) by which certain components of the extra-c e l l u l a r matrix promote s p e c i f i c stages of cytodifferentiation i s not clear, p a r t i c u l a r l y since i n a number of cases, simple addition of p u r i f i e d matrix components such as chondroitin sulphate or c o l l -agen does not sustain cy t o d i f f e r e n t i a t i o n (14 ,34) . However, components of the extracellular matrix could affect c e l l u l a r f u n c t i o n i i n d i r e c t l y by providing a microenvironment of variable adhesivenessfeand'orient-ation due to their i o n i c and fibrous nature (43,49}50)isthereby mastering c e l l configuration (5>D), c e l l - c e l l attachments^) ^'T^yie&£tec1&£yi±ffl£uea-cing c e l l surface properties. Certainly, i n the case of corneal epithelium development, direct contact with collagen substratum i s necessary, suggesting that a membrane interaction (8) i s required for the inductive event i n this tissue. Neither collagen, chondroitin sulphate or hyaluronate are tissue s p e c i f i c or unique to s p e c i f i c stages during embryonic development. The gross chemical composition of the matrix components are similar i n the embryo and adult even though at precise stages, certain tissues only, w i l l react to their presence ( 8 , 4 2 - 4 5 ) . The ubiquitous nature of these components suggests that tissue s p e c i f i c i t y i s a property of the reacting c e l l , i n turn implying that the development of tissue s p e c i f i c i t y i s v i a another mechanism(s). Nevertheless, although current techniques do not discern differences i n gross molecular composition, t h i s does not mean, of course, that they do not exi s t : minute structural d i f f -erences could conceivably lend tissue s p e c i f i c i t y to extr a c e l l u l a r matrix molecules. In f a c t , recent work (46=48) suggests that a 7 tissue s p e c i f i c i t y i n the three dimensional construction of matrix components exists which might provide a precise architectural effect necessary to s p e c i f i c a l l y a l t e r tissues during c r i t i c a l develop-mental stages. Such differences could be genetic or epigenetic (46). Current work by Toole and coworkers i n t h i s area should provide answers to these interesting questions i n the near future. Pre In the i n i t i a l stages of the work reported here, methods were developed to propagate normal adult rat adrenal c o r t i c a l c e l l s i n monolayer culture without the addition of adrenocorticotrophic hormone (53,54) and subsequent studies suggested that certain extracellular factors including e x t r a c e l l u l a r matrix components influenced c e l l phenotypic expression, possibly by a l t e r i n g growth patterns. Previous investigations of the role of ex t r a c e l l u l a r mat-e r i a l i n the adult mammal had been primarily limited to the import-ance of matrix components i n determining rheological properties of connective tissues and examining the relationship between a l t e r a t i o n of matrix composition and disruption of these properties i n conn-ective tissue diseases (49). However, the a b i l i t y of extracellular matrix to influence phenotypic expression of adrenal c o r t i c a l c e l l s suggested that matrix components can, i n f a c t , exert an influence on adult c e l l function. Further studies attempted to delineatetthe mechanism(s) by which the ext r a c e l l u l a r matrix as w e l l as serum factors controlled phenotypic expression of adrenal c o r t i c a l c e l l s in vitro. P a r t i c u l a r emphasis has been placed on the i n t e r -relationships among c e l l u l a r form, c e l l - t o - c e l l contact and c e l l function with the ultimate hope of becoming aware of the extra-8 cellular factors that might influence adrenal cortical phenotypic expression in vivo other than the classical trophic hormone interactions. MATERIALS AND METHODS I. C e l l Culture A. Primary cultures Adult male Fischer r a t s , about 3 months old, were k i l l e d by immersion i n an atmosphere of CO2 or ether. The adrenal glands were removed aseptically and minced i n culture medium. Each gland was ex-planted into a 35x10 mm tissue culture dish (Falcon p l a s t i c s ) . Diss-ociation of the glands by trypsin was avoided because preliminary tests showed adverse effects on culture growth. I n i t i a l experiments (53) had shown that higher concentrations of f e t a l calf serum (FCS) supported the most extensive c e l l migration and growth while l i t t l e or no migration was observed i n primary cultur grown with horse serum (HS) supplements. Therefore a l l primary cultur were grown i n Waymouth's medium MB 752/1 (5§) supplemented with 25% FCS, fl00^units/ml p e n i c i l l i n and lOOyg/ml streptomycin (FCS-medium) and were incubated at 37°C i n a humidified atmosphere containing 5% C0 2. The effect of different batches of serum (Gibco and Reheis) as well as the effect of the age,strain and sex of rats on the extent and morphology of primary outgrowths were examined also using the above culture techniques. B. Secondary cultures The effects of three variables on the morphology, growth rate and steroid production of c e l l s propagated from primary cultures obtained from adult male Fischer rats were compared: 1. the c e l l density i n primary culture at the time of subculture; 2. the method of c e l l dissociation; and 3. the serum supplement. Cells > t were subcultured from (1) a confluent state i n one groups of cultures and from a nonconfluent state i n another group of cultures. Three dissociation (2) methods and several serum supplements (3) were compared within each group (1): the influence of the extent of c e l l dissociation by enzymatic or non-enzymatic methods upon subse-quent culture morphology of monolayer fragments obtained from confluent primary cultures was assessed. Monolayer fragments were suspended i n either trypsin or HS-medium and pipetted for 30 sec, lmin or 2 min. Examination of the resulting suspension showed that after 30 sec of pipetting, large c e l l groups had remained i n t a c t , after 1 min of pipetting only small monolayer fragments werenobserved and after 2 min of pipetting, only single c e l l s were observed. Cells were also dissociated mechanically by cutting the monolayers into fragments and then scraping monolayers from growth surface with the edge of a si l i c o n e stopper. A l l c e l l suspensions were then centrifuged and resuspended i n fresh medium. Cells were then plated (1:2d'diitu.Mon) i n 1.5 ml/dish of medium MB 752/1 with 10,15,25* and 35% FCS or 3,5 and 10% HS. During the course of these experiments i t was found that the morphology and steroid production of adrenal c o r t i c a l c e l l s i n sec-ondary culture varied when grown i n medium supplemented with d i f f -erent l o t s of HS (Table 1). In l o t NAB 201189, c e l l s attained an e p i t h e l i a l - l i k e morphology with 10% HS supplement but were f i b r o b l a s t -l i k e with a 25% supplement. In l o t Gibco A942211, c e l l s were epith-e l i a l - l i k e with 1-3% HS but fibrobla-st-like with 5-10% supplements. In 3 other l o t s of horse serum i n concentrations ranging from 3-10%, a f i b r o b l a s t - l i k e morphology was always observed while i n 2 other l o t s , an intermediate morphology was observed at these concentrations. Table 1 The Effect of Different Lots of Horse Serum on the A b i l i t y of Secondary Monolayer Fragments, Obtained From Confluent Primary Cultures, To Attain an E p i t h e l i a l Morphology. Supplier Sera Lot % Serum Supplement Culture Morphology Steroid Production3 ug/24h /10 6 cells;.. North American 201129 25 f i b r o b l a s t - l i k e not assayed B i o l o g i c a l Co. 10 e p i t h e l i a l 4.79 ± 0.51 203345 10 f i b r o b l a s t - l i k e trace 5 f i b r o b l a s t - l i k e trace 3 f i b r o b l a s t - l i k e trace K.C. Bi o l o g i c a l Inc. 26039 3 f i b r o b l a s t - l i k e trace Microbiological 88182 3 e p i t h e l i a l , no l i p i d Associates G1BC0 A942211 10 f i b r o b l a s t - l i k e trace 5 e p i t h e l i a l to intermediate 1.17 ± .13 3 e p i t h e l i a l , l i p i d inclusions 0.97 ± .35 1 e p i t h e l i a l , l i p i d inclusions trace -. C942216 5 e p i t h e l i a l to intermediate 1.4+ 1.' 0: • 3 e p i t h e l i a l to intermediate 2.9 ± 2.0 E040118 5 f i b r o b l a s t - l i k e trace 3 f i b r o b l a s t - l i k e trace values represent the mean±S.E.M. of 4 cultures. 12 Generally, c e l l groups exhibiting a f i b r o b l a s t - l i k e morphology (i e . when maintained i n HS l o t s NBA 203345, KCB 26039 and Gibco E040118) produced only trace amounts of fluorogenic steroid (Table 1). C e l l groups that were intermediate i n morphology ( i e . maintained i n HS l o t s Gibco C942216 and A9'42<112 at 5% supplement) produced up to 3.0)jg of steroid/10 6 cells/24 hsbut production was extremely variable. C e l l groups growrin i n 1-3% HS, l o t Gibco A942211 were e p i t h e l i a l - l i k e and contained numerous l i p i d inclusions but steroid production was consistently lower than c e l l s of similar morphology maintained i n HS l o t NAB 201189. Furthermore, steroid production was even lower at concentrations of 1% although the morphology remained e p i t h e l i a l - l i k e at t h i s serum concentration and c e l l s app-eared to be healthy. According to Sato et al. (25$) , steroid production by Y - l adrenal c o r t i c a l c e l l s i s increased by addition of up to lmg/ml of bovine serum albumin which acts as a steroid carrier and t h e o r e t i c a l l y prevents negative feedback i n h i b i t i o n of steroid production (25.7)). Therefore,, the effect of bovine serum albumin (fraction V, Fisher Chemicals) on fluorogenic steroid production was determined. 0.05-0.2 mg/ml of BSA was added to c e l l s at the time of subculture. Subsequently, monolayers were incubated with fresh medium for 24 h without BSA and steroid determined using acid fluorometry (see materials and methods, section III-D). Steroid production i n HS l o t Gibco A942211 could be increased from lug/10 6 cells/24 h to about 3-4ug/106 cells/24 h i n the presence of BSA at concentrations of 0.2mg/ml. Addition of BSA did not a l t e r culture morphology (Table 2). The steroid production by adrenal f i b r o b l a s t - l i k e c e l l s maintained i n FCS-medium did not vary with the serum l o t but remained consistently low. Furthermore, the addition Table 2 Influence of the Addition of Bovine Serum Alumin (Fraction V) On Fluorogenic Steroid Production of Adrenal Cultures. Growth Conditions Culture Morphology Bovine Serum Albumin mg/ml Steroid Production 1 ug/24hr/106 c e l l s medium supplemented with 3% horse serum (GIBCO l o t //A942211) medium supplemented with 25% FCS e p i t h e l i a l with l i p i d inclusions f i b r o b l a s t - l i k e 0 0.2 0 0.2 0.95 ± 0.35 2.48 ± 1.80 0.01 ± 0.002 0.01 ± 0.003 values represent the mean±ES.E.M. of 8 cultures. of BSA did not elevate steroid production. Therefore, after the o r i g i n a l l o t of HS (NAB 201189) was depleted, Gibco l o t A942211 at a concentration of 3% and supplemented with 0.2mg/ml of BSA was used as the HS-medium. The influence of the s t r a i n , sex and age of rat (see IA) upon secondary culture morphology was assessed i n secondary cultures prepared as described i n B. C. S e r i a l l y passaged cultures a. mor_phol_og_y The a b i l i t y of subsequent passages of adrenal e p i t h e l i a l - l i k e and f i b r o b l a s t - l i k e c e l l s to r e t a i n their morphology i n the culture conditions described above was ascertained by (a) mechanically sub-culturing monolayer fragments of stationary secondary, t e r t i a r y and i quaternary culture's of c e l l s i n 10% HS-medium into 25% FCS-medium and (b) mechanically subculturing monolayer fragments of confluent secondary t e r t i a r y and quaternary cultures propagated i n FCS-medium onto 10% HS-medium. b. l_ifespa.n_ The lif e s p a n :lof adrenal c e l l s in vitro was compared between cultures propagated by weekly t r y p t i c dissociation and grown i n FCS-medium and cultures propagated by weekly mechanical dissociation and grown i n HS-medium. In both groups, c e l l s were diluted 1:2 at each passage. D. C e l l number Unless indicated otherwise, the c e l l number i n cultures was estimated by preparing a single c e l l suspension by t r y p t i c digestion of monolayers and counting i n a hemacytometer. A minimum of 200 c e l l s were counted per c u l t u r e d i s h . E . Muscle f a s c i a c u l t u r e s Cultures of connective t i s s u e f i b r o b l a s t s from muscle f a s c i a were grown as c o n t r o l s i n an i d e n t i c a l manner to the adrenal c e l l s . 16 I I . Histochemistry A. Enzymes and l i p i d Cultures were stained for A5-3g-hydroxysteroid dehydrogenase by the method of Levy et al. (58) with s l i g h t modifications (appendix I) and for glucose-6-phosphate dehydrogenase using the method of Hess et al. (59). L i p i d was demonstrated with o i l red 0. B. Carbohydrate The nature of mucin present i n e x t r a c e l l u l a r matrix produced by adrenal c o r t i c a l c e l l s i n FCS-medium and routinely demonstrated by metachromatic staining with toluidine blue at pH 2.5, was further investigated as outlined i n Table 3. Staining was conducted as outlined by Culling (60). A l l chemicals were purchased from Fisher Chemicals. 17 Table 3. SIMPLIFIED CHART FOR MUCIN IDENTIFICATION Type of mucin (reactive group) Neutral Mucin XI:2 glycol) PAS/Alcian blue pH 2.5 Red B Alcian blue pH 1.0 Negative KOH-acid hyd-r o l y s i s -Alcian blue, pH 2.5 D Hyalase-Alcian blue 1 pH 2.5 Acid Mucin (COOH) Blue-Purple Negative Sulphated Mucin (0S03H) Blue-Purple Blue Connective Tissue Mucin Blue-Puxple Blue or Negative Blue Negative or reduced E p i t h e l i a l Mucin Blue-Purple Blue or Negative or Blue Negative reduced "''Ovine t e s t i c u l a r hyaluronidase (Sigma Chemicals, Mo.) I I I . Steroid Determinations 18 A. Steroids Non-radioactive steroids, pregnenolone, pregn-5-ene-3g,20a-diol, 20a-hydroxy-pregn-4-en-3-one, deoxycorticosterone and corticosterone were obtained from Steraloids, Pawling, N.Y. [4- 1^C]pregnenolone (52.8 mCi/mmol) , [4- 1 4C]progesterone (57.3 mCi/mmol) , [4-1'tC] c o r t i -costerone (57.3 mCi/mmol) , [4- 1 1 +C]deoxycorticosterone (54.3 mCi/mmol) and [1,2-3H(N)]20a-hydroxypregn-4-en-3-one (40 mCi/mmol) were pur-chased from New England Nuclear Corporation, Boston, Mass. Before use, they were chromatographed i n the system PPC 1 (see below). B. Liquid scintiJilationn counting Radioactivity was determined i n toluene containing 2,5-diphenyl-oxazole (12 g/L) and 1,4-bis-[2-5(phenyloxazolyl)]-benzene (300mg/L) using a Packard model 526 a l i q u i d s c i n t i l l a t i o n spectrophotometer. Counting ef f i c i e n c y was 86% for 11+C and 35% for 3H. C. Chromatography a. P_ap_er_ c^hr_om^at_o^r^phy_(^jjendlx_II_) System PPC 1: Whatman No. 1 paper was impregnated with propylene gly c o l and developed with l i g h t petroleum, (b.p. 63-75°C) for 24 h. System PPC 2: Whatman No. 1 paper was impregnated with prop-ylene gl y c o l and developed with benzene:hexane (1:1, v/v) for 24 h. b. thJ:n^laye_r_chroma.t^gr_ap_h^ systems_ ( S i l i c a Gel G plates) TLC 1: Benzene:ethyl acetate (3:1, v/v). TLC 2: Chloroform:ethyl acetate (1:50, v/v). Inert c a r r i e r steroids were located either under u.v. l i g h t or by phosphomolybdate (appendix I I I ) . Radioactive metabolites and standards were located using autoradiography as described by J e l l i n c k 19 and Goudy (61). D. Incubation, i s o l a t i o n and i d e n t i f i c a t i o n of the radiometabolites a. j^ econda.ry_ c_uj.tu_re_s_de_r^ved_fr_om a. 1_:2_ spl_it_ ^t_the_ti_me ^ f_subc:ul_ture_ In order to assess whether adrenal c e l l s described here could form intermediates i n the adrenocorticoid biosynthetic pathway, [4- l l tC]pregnen-olone was added as a substrate and the metabolites isolated and i d e n t i f i e d . i ) confluent cultures Stationary secondary cultures were incubated for 8 h with approxi-mately 200,000 d.p.m. of radioactive steroid i n 2 ml medium per culture dish. After incubation, the c e l l s were dissociated with 0.12% trypsin and counted i n a hemacytometer. The incubation mixture was homogenized i n a Teflon blender and extracted twice with dichloromethane. The dichloro-methane extract was taken to dryness under nitrogen, admixed with lOOug of unlabelled c a r r i e r i n methanol and then sequentially chromatographed i n the systems PPC 1, PPC 2, TLC 1, TLC 2 and after acetylation (appendix III) i n TLC I. Of the metabolites i s o l a t e d , pregn-5-en-3820ct-diol, 20ct-hydroxy-pregn-4-en-3-one, deoxycorticosterone and corticosterone were i d e n t i f i e d by c o - c r y s t a l l i z a t i o n with authentic c a r r i e r steroids. After 3 successive c r y s t a l l i z a t i o n s , the s p e c i f i c a c t i v i t y of the c r y s t a l crops and of the l a s t mother liquors remained constant within an error of no more than ±3.10% (coef. of variation) (Table 4 ) . Samples were read on a Tracor mt-22-gas chromatograph. Since most of the metabolites were d i f f i c u l t to c r y s t a l l i z e from solvent pairs other than methanol/water, this was used consistently. Axelrod et al. (62) have shown that the use of one solvent pair to obtain radiochemical purity i s j u s t i f i a b l e . i i ) non-confluent cultures Determination of [4- 1^Cjpregnenolone metabolism of monolayers at different stages of confluence was accomplished as follows: a 20 Table 4 Rec r y s t a l l i z a t i o n Data 1 of Steroids Metabolized by Adrenal E p i t h e l i a l - l i k e and F i b r o b l a s t - l i k e Cells From [ 4-11+C] pregnenolone Specific A c t i v i t y 2 Steroid l a s t 1st Crystal 2nd Crystal 3rd Crystal mother Mean liquor A. Adrenal E p i t h e l i a l - l i k e Cells pregnenolone 1157 1144 1168 1135 1151 pregn-5-ene-3g,20a-diol 1425 1409 1407 1436 1419 20a-hydroxy-pregn-4-en-3-one 4821 5010 4971 4918 4930 deoxycorticosterone 3090 3250 3195 3150 3171 corticosterone 3 3140 3000 2980 3010 3032 B. Adrenal F i b r o b l a s t - l i k e Cells pregnenolone 4236 4444 4138 4203 4255 pregn-5-ene-3g,20a-diol 1009 1004 1006 1035 1013 20a-hydroxy, pregn-4-en- 3-one 576 582 545 580 570 deoxycorticosterone 3 1000 958 968 970 974 Values represent pooled metabolites from 10-20 cultures. The s p e c i f i c a c t i v i t y of the l a s t three crystals and the l a s t mother liquor are given. Values are expressed as dpm/mg authentic steroid. The solvent used for r e c r y s t a l l i z a t i o n was MEOH/H2O. Steroids metabolized from [4-lt+C ]pregnenolone after the addition of ACTH (200 my per P e t r i dish/day.) 21 suspension of c e l l s trypsinized from primary monolayers was diluted 1:2 and [4- 1 1 +C]pregnenolone metabolism assessed as described above at 1,3,5 and 7 days after plating. b. ^e£ondary_ ilu—tu_r—§ derived from JLej3S_than z\u_9£^is_ [ 4-11+C] pregnenolone metabolism of micropopulations of f ibroblast-l i k e adrenal c e l l s was accomplished as follows: primary cultures were dissociated with 0.12% trypsin as described i n c e l l culture techniques and resuspended i n 10 ml of fresh medium; 0.1 ml o f - t h i s suspension was added to £ivemiLr<7 Gml.lL'inbr6cwelilsa.(?Ealeon Pl'astics)Y Cells were allowed to s e t t l e and then a p l a s t i c cylinder of 5mm diameter was placed over a small c e l l group, usually of 10-40 c e l l s . The cylinder was maintained i n place u n t i l c e l l s within the enclosed area were confluent. The remaining c e l l s outside the cylinder were scraped from the surface and pipetted off. The cylinder was removed and c e l l s allowed to grow to confluence. [4- 1^C]pregnenolone metabolism was determined as described above except that 100,000 d.p.m. of [4- l l tC]-pregnenolone i n 1 ml of medium was added to the monolayers so that substrate concentration was similar to that i n 35x10 mm tissue culture dishes. E. Isolation and?identification of endogenous steroid Cultures were/incubated with 2 ml of fresh medium for 24 h. The supernatant medium of each culture dish was then extracted with 10 ml of dichloroihethane. After removal of the aqueous phase and f i l -t r a t i o n through Whatman No. 1 f i l t e r paper, the extract was shaken with 2 ml.of 65% (v/v) ethanolic sulphuric acid. After 60 min, the fluorescence of the sulphuric acid layer was determined with an Aminco-Bowman spectrophotofluorometer using an excitation wavelength 22 of 470 nm and an emmisslon wavelength of 525 nm. Corticosterone was used as the reference standard. To i d e n t i f y the major steroids produced endogenously by the c e l l s , the extracts of 8-10 incubates of each c e l l type were pooled. To each pool were added 0.06jjg each of [4- 1 1 +Cfprogesterone, [4- l l +C]-pregnenolone, [4- 1^C]deoxycorticosterone and [1,2-3H(N)]20a-hydroxy-pregn-4-en-3-one. Each mixture was chromatographed i n the system PPC 1 and then i n the system TLC 1. Radioactive areas, located by autoradiography were eluted with ethanol. The f i n a l eluates were dried under N 2» redissolved i n dichloromethane, extracted with 65% ethanolic sulphuric acid and the fluorescence determined as above. Values were adjusted for loss of steroid during the chromatography and elution procedure as assessed by the loss of radioactive standard. IV. Microscopy A. Light microscopyy Cultures i n s i t u were fixedd with 2.5% glutaraldehyde i n phosphat buffer at pH 7.4 and stained with 2% t o l u i d i n e blue at pH 2.5 ( i n 3% ac e t i c acid) for metachromasia. C e l l s were photographed on a Zeiss photomicroscope when stained or photographed a l i v e with phase optics on a Wild inverted photomicroscope. Materials prepared f o r electron microscopy were also examined by l i g h t microscopy. Epon sections, 0.5-1.0 micron-thick were cut with glass knives on a Reichert ultramicrotome. The sections were transferred to glass s l i d e s with a f i n e wire loop, heat fixeddontb the s l i d e s and stained with 1% t o l u i d i n e blue i n 1% borax ( 63 ). B. Electron microscopy a. t_r^n^m^s^.l^n_el_ec^tr_on microscopy Adrenal c o r t i c a l c e l l s were grown to confluency i n 35x10 mm ti s s u e culture dishes. Following incubation i n one of the following: 1) basal medium (Waymouth's medium supplemented with either HS or FCS) 2) medium containing 100 mU/ml ACTH f o r 1 day 3) medium containing 100 mU/ml ACTH for 3 days 4) Tnmedium containing 100 mU/ml ACTH for 5 days the cultures, i n s i t u , were washed gently with Hanks' balanced s a l t s o l u t i o n , f i x e d i n cold glutaraldehyde i n phosphate buffer followed by post f i x a t i o n i n 1% osmium tetroxide i n M i l l o n i g ' s buffer f o r 30 minutes and again washed twice with buffer. The cultures were dehydrated through a graded series of ethanol and then scraped o f f the growth surface, suspended i n 100% ethanol followed by propylene oxide and i n f i l t r a t e d with epon according to routine procedure (append IV). Then, the monolayer fragments were transferred with a tooth-pick to f l a t embedding dishes - f i l l e d with..eporii.and .polymerized '. (37°C for 12 h and 56°C for 24hh). Appropriate areas of the embedd-ed cultures were selected by l i g h t microscopy, cut out and mounted on blocks for sectioning. Thin sections were cut with glass knives and with a Du Pont diamond knife on a Reichert ultramicrotome, moun-ted on carbon coated copper grids, stained with uranyl acetate (64) and lead c i t r a t e (65) and examined with either an Hitachi HS-7S electron microscope or a Zeiss 110 electron microscope. b. scjmning_el_ectr_on. microsc.oj£y_of_ un_treat_ed ,_c h^ a rg ed_j_ nit_rjLc_acid_ e^tche^d_and_bru^he_d_grjDwth surf aces To ffiacilitiateee handling and manipulation, areas of randomly selected growth surfaces were cut out and mounted on aluminums spec-imen stubs with adhesive copper tape adfixed to the aluminum surface with S i l v e r Dag mounting medium. To render the specimen conductive, the surfaces were coated with 150 X-thick layer of gold i n a vacuum evaporator. The surface topography of the growth surfaces was ex-amined i n a Cambridge stereoscan scanning electron microscope. c. t_imel_ap_se cinemic_ro_gr_ap_hy_ Cultures were grown i n 25 cn? tissue culture flasks and gassed with CO2 to obtain an equilibrium of 5% C02/95% a i r , as assessed by the pH of the medium. The cultures were maintained at 37°C by a Sage a i r curtain incubator and the temperature was monitored by a thermometer next to the cultures. The c e l l s were photographed on a Wild inverted microscope using phase optics (lOx objective) with a cine-Kodak special I I camera (Kodak, U.S.A.). The entire assembly of camera, camera stand and microscope were mounted onto an a n t i -v i b r a t i o n plate (Wild). Recordings were made on 16 mm Plus X neg-ative f i l m (Eastman Kodak #7231, ASA 64). Interval (1 frame/min) and exposure time (0.1 sec) were controlled by an Embdecco time lapse drive control (model 783, T - l , Electro-mechanical Develop-ment Co., Houston, Texas). Film was subsequently analyzed on a Zeiss moviscope, and c e l l movement assessed by tracing the displace-ment of c e l l nuclei as described by Garrod and Steinberg (66) of a t o t a l of 10 randomly chosen c e l l s within each experimental group. V. Experimental Manipulation of Secondary Cultures 26 A. ACTH and LH The effect of adrenocorticotrophin (ACTH) and of l u t e i n i z i n g hormone (LH) on the endogenous production of fluorogenic steroid and on [4- 1^C]preg-nenolone metabolism by adrenal c e l l s was examined by the daily replacement of old medium with 2 ml of fresh medium containing 200 mU ACTH (porcine, 88 i.u./mg, Sigma Chemicals) for 1, 3 and 5 days before steroid determina-t i o n . The stimulatory effect of ACTH on [4- 1^C]pregnenolone metabolism was s t a t i s t i c a l l y tested using a two-tailed T test. Cultures were photo-graphed during ACTH treatment using phase optics on a Wild inverted micro-scope. The effect of ACTH on c e l l d i v i s i o n was estimated by counting the number of metaphase plates per 1000 c e l l s i n 8 cultures of control and ACTH-treated (1,3 'and 5 days) monolayers. The effect of adding 200 mU/2 ml medium of LH (Bovine, NIH, Bethesda Md) on endogenous steroid production was examined as described for ACTH. Cultures of connective tissue f i b r o -b l a s t s , taken from muscle f a s c i a , were treated i n an i d e n t i c a l manner and served as controls. B. Exchange of serum supplements The effects of substituting medium supplemented with 25% f e t a l . cal f serum (FCS-medium) for medium supplemented with 3% horse serum and bovine serum albumin (HS-medium) were examined as follows: secondary monolayers of adrenal e p i t h e l i a l - l i k e c e l l s were obtained by mechanically dissociating the confluent primary outgrowths by f i r s t cutting them with a scalpel and then scraping them off the p l a s t i c with the edge of a s i l i c o n e stopper and plating the resulting mono-I layer fragments into HS-medium. Cells were diluted 1:2 at the time of subculture. Three days after subculture, when highly d i f f e r e n t -iated e p i t h e l i a l islands had formed, HS-medium was replaced with lh i , •: , Mill l l lMfCl l l l l l l i l lN FCS-medium. Using acid fluorometry (67) , endogenous steroid prod-uction was assayed at 0,8,20,40 and 48 h after the addition of FCS-medium. Sample cultures from each i n t e r v a l were fixed with 2.5% glutaraldehyde and stained with aqueous toluidine blue at pH 2.5 to detect metachromasia. The modulation of e p i t h e l i a l - l i k e c e l l s to a f i b r o b l a s t - l i k e form i n the presence of f e t a l c a l f serum was followed for 48 h with time lapse cinemicrography i n p a r a l l e l c u l t -ures maintained i n tissue culture f l a s k s . M i t o t i c a c t i v i t y was estimated by counting mitotic figures i n a t o t a l of 8,000 c e l l s (6 cultures) and expressing the value as mitotic figures per 1000 c e l l s . The effect of substituting HS-medium for FCS-medium was examined as follows: FCS-medium i n which 5 day old secondary cultures of adrenal f i b r o b l a s t - l i k e c e l l s were grown was replaced with HS-medium for 3 days. Cultures were subsequently incubated for a further 24 h with fresh HS-medium which was then analyzed for fluorogenic steroid. Sample cultures were stained with toluidine blue as described i n microscopic techniques (section IV,A) and the remaining monolayers were trypsinized and counted i n a hemacytometer. C. 6-d iazo-5-oxo-L-nor1eu cine (DON) Monolayer fragments obtained mechanically from confluent primary cultures were plated into HS-medium. On the third day after sub-culture, the following treatments were carried out: 1. HS-medium was replaced with fresh HS-medium for a 24 h c o l l e c t i o n of steroid and subsequently assayed for steroid using acid f l u o r -ometry; 2. 50?if)ig DON/ml was added to monolayers for 24 h; 3. HS-medium was replaced with 10% FCS-medium for 24 h; 28 4. HS-medium was replaced with 10% FCS-medium plus 5-100ug DON/ml for 24 h; 5. HS-medium was replaced with 10% FCS-medium plus 50yg DON/ml and either lmg L-glutamine/ml (Lyophylyzed, Gibco), O.lmg glucosamine-6-phosphate/ml (Sigma Chemicals) or O.lmg D-glucosamine/ml (Sigma Chemicals) for 24 h. A l l monolayers except 1. were subsequently incubated with fresh HS-medium containing no additives for 24 h and steroid produced during this time was assayed by acid fluorometry. In the case of 4, the steroid production of monolayers incubated with 50ug DON/ml only was examined. In a l l treatments (1-5), sample cultures were stained with 2% toluidine blue at pH 2.5 for detection of metachromatic e x t r a c e l l u l a r matrix and the remaining monolayers were trypsinized and counted i n a hemacytometer. Monolayer fragments prepared and grown as described above were prepared for time lapse cinemicrography as described i n section (IV,c). D. Hyaluronic acid and chondroitin sulphate Monolayer fragments obtained mechanically from confluent primary cultures were plated into HS-medium. On the t h i r d day .after subculture, hyaluronic acid (human umbilical cord, grade 1, Sigma Chemicals ; (approximately 1 x 10 6 M.W.), and p u r i f i e d hyaluronic acid, g i f t of Dr. B. Toolei or chondroitin sulphate (shark and whale cartilage, mixed isomers, Sigma Chemicals.,approximately 4 x 10^ M.W.) were added to cultures i n HS-medium i n concentrations ranging from 50-500yg-/ml for a t o t a l of 24 h. A l l monolayers were subsequently incubated with fresh HS-medium without added glycosaminoglycans for 24 h and the steroid collected during this time was analyzed using acid fluorometry and compared with the steroid production ofO'cVontro'l cultures of untreated e p i t h e l i a l - l i k e monolayers. Sample cultures were stained with t o l u i d i n e blue as described previously while remaining monolayers were tr y p s i n i z e d and counted i n a hemacytometer. E. A l t e r a t i o n of growth surfaces a. p_repa.ra.tiori £f_growth surfaces i ) sulphonated surfaces Sulphonated growth surfaces were prepared by incubating 35x10 mm t i s s u e culture dishes with 2 ml of concentrated sulphuric acid (reagent grade, Fisher Chemicals) at 55°C for 1,3,5,15,30 min and 1,2,4, and 10 h. The dishes were washed for 24 h i n running water, rinsed i n d i s t i l l e d water four times and s t e r i l i z e d under u.v. l i g h t . The degree of sulphonation of the growth surfaces was assayed by dye binding ( 68 ): the surfaces were eq u i l i b r a t e d with a s o l u t i o n of t o l u i d i n e blue, 2.5%, pH 2.5, and washed with d i s t i l l e d water for 10 min to remove excess dye. The remaining dye was eluted with acid methanol (HC1:ME0H, 1:5, v/v) and measured c o l o r i m e t r i c a l l y on a Coleman Junior Spectrophotometer at 640 my. The amount of bound t o l u i d i n e blue increased from 0.005mg on untreated ti s s u e culture dishes to 0.11 mg on sulphonated dishes (Table 20). i i ) p o l y l y s i n e treated surfaces P o l y l y s i n e treated surfaces were prepared as described by Le-tourneau ( 69). B r i e f l y , 2 ml of a lmg/ml solution of poly-D-lysine hydrobromide (type VTI-B, Sigma Chemicals) i n borate buffer (0.1 mM, pH 8.4) was added to 35x1*0 mm tis s u e culture dishes for 24 h, rinsed with s t e r i l e d i s t i l l e d water and l e f t i n d i s t i l l e d s t e r i l e water f o r 5 days, changing water every day. Dishes were then r i n s e d 3 times i n Hanks' balanced s a l t s o l u t i o n p r i o r to use. i i i ) brushed surfaces . 35x10 mm tissue culture dishes were brushed for several minutes with a Lofstrand c i r c u l a r rotating glass washer mounted with a s t i f f brush. Dishes were then rinsed i n running tap water for 2 hours, rinsed 3 times with s t e r i l e d i s t i l l e d water and then rinsed 3 times with Hanks' balanced s a l t solution prior to use. iv) n i t r i c acid treated surfaces N i t r i c acid treated surfaces were prepared by incubating 35x10 mm tissue culture dishes with 2 ml of concentrated n i t r i c acid (reagent grade, Fisher Chemicals) at 55°C for 30 minutes. The dishes were then washed for 24 h i n running water, rinsed i n d i s t i l l e d water four times and s t e r i l i z e d under u.v. l i g h t . Dye binding of sample dishes was assayed as described for sulphonated surfaces. hi ra_te_ of_c e l l _ £itt_aj:hmerit The rate of attachment of monolayer fragments to treated growth surfaces was estimated as follows: monolayer fragments obtained mechanically from confluent primary cultures were suspended i n (a) HS-medium; (b) Waymouth medium only; or (c) Hanks' balanced salt salfetsq{Lutii;on ,.?£fid . ec]ua<L. amdun^aofasc^gSfisiifonSwgMngS^Scle' «• " • to p e t r i dishes, tissue culture dishes, sulphonated, polylysine treated, n i t r i c acid treated and brushed fiissueeculture surfaces. Fragments were allowed to s e t t l e for 5 minutes and then medium and unattached fragments were removed by inversion. The number of frag-ments l e f t attached to growth surfaces was counted as were the un-attached fragments removed with the medium. Rate of attachment was calculated as percent attached fragments/5 min. The settlement of cold-treated monolayer fragments wassexamined as follows: monolayers fragments obtained as described above were suspended i n Waymouth's medium without serum and incubated at 4UC for 1 h. Attachment was then assessed as described above. The rate of attachment of fixed c e l l s was examined as follows: monolayer fragments obtained as described above were suspended i n Waymouth's medium and an equal volume of cold 2.5% glutaraldehyde i n phosphate buffer at pH 7.4 was added while mixing vigorouslyv :, as described by Yip and Auersperg ( 70). The mixture was l e f t at 4°C for s.h. Fixed fragments were then centrifuged, glutaraldehyde containing medium was removed and fresh medium added. Monolayer fragments were washed i n t h i s manner several times, to remove a l l traces of free glutaraldehyde. Monolayer fragments were allowed to equilibrate to room temperature for { h, the rate of attachment of fixed c e l l s was then assessed as described above. FJ? Mechanical dissociation Monolayer fragments were obtained mechanically from confluent primary cultures and suspended i n HS-medium. The suspension was pip-etted vigorously for several minutes and then centrifuged and resus-pended i n fresh medium. The resulting single c e l l s were plated i n 1:2 d i l u t i o n i n 35x10 mm tissue culture dishes. Three days l a t e r , fresh HS-medium was added, collected 24 h l a t e r and analyzed for steroid using acid fluorometry. G. Colcemid and lumicolchicine a. addit_ion_o_f ^olcemid_tc> j:ul_tures Colcemid (Gibco) was prepared fresh as a 10-1* M stock solution i n s t e r i l e d i s t i l l e d water and 0.01 ml of t h i s added to monolayers 3 and 5 days after plating ( f i n a l concentration, 10 6M) for a t o t a l of 8 and 48 h. Medium containing colcemid was then replaced with f r e s h medium minus the a l k a l o i d and cultures were either incubated with [ 4- 1 1 +C] pregnenolone for 8 h or with medium for 24 h and l a t e r ana-lyzed for fluorogenic s t e r o i d , b. jjrejia_rati.ori of_lumicol.chic_ine Lumicolchicine was prepared according to the method of Miz e l and Wilson (7>1>). B r i e f l y , a 10 1+M solut ion of c o l c h i c i n e was prepared i n ethanol. The s o l u t i o n was placed i n a glass cuvette and i r r a d i a t e d for up to 5 h at 25°C by a Blak Ray long-wave u l t r a v i o l e t lamp (UVL-22 U l t r a v i o l e t Products Inc., San G a b r i e l , C a l i f o r n i a ) . Conversion of c o l c h i c i n e to lumicolchicine was followed by decreasing absorb-ance at 350 my and increasing absorbance at 267 my. The reaction was judged to be complete when the solution did not absorb at 350 my and absorption at 267 my reached and maintained a plateau (Bigg . 33 FIGURET. Conversion of colchicine to lumicolchicine in the presence of ultraviolet light. ABSORBANCE AT 267 mu -i 1 1 r 0 I 3 HOURS OF IRRADIATION RESULTS 34 I. Growth Conditions A. Primary cultures Primary adrenal cultures obtained from male Fischer rats of about 2-3 months of age and grown i n Waymouth's medium with 25% FCS, consisted of predominately f i b r o b l a s t - l i k e c e l l s (Fig. la) interspersed with occasional groups of l i p i d - l a d e n and e p i t h e l i a l c e l l s (Fig. l a and b) similar to those described by Kahri (72) and Armato and Nussdorfer (73) as highly functional. Growth of c o r t i c a l explants from 2-3 month old Fischer male rats i n different f e t a l c a l f serum l o t s had no noticeable effect on primary culture morphology. However, outgrowth as well as culture morphology did vary with the age, sex and s t r a i n of rat (Table 5). Monolayers of explants from 4-7 week old male rats contained fewer l i p i d and e p i t h e l i a l c e l l s than those from 2-3 month old males. I n i t i a l outgrowth and confluence of monolayers were reached e a r l i e r i n the explants from younger ra t s . Explants from 7 week old females showed similar outgrowth rate and culture morphol-ogy to males of that age although, from rat to r a t , outgrowth was more variable. However, no outgrowths were observed i n c o r t i c a l explants from adrenals of 10 week old females. C e l l outgrowths from male Wistar adrenal c o r t i c a l explants were en t i r e l y f i b r o b l a s t - l i k e , regard-less of age (4-12 weeks). These l a s t results are sim i l a r to those reported by O'Hare and N e v i l l e (74) using the Wistar s t r a i n . B. Secondary cultures The morphology of secondary and subsequent cultures obtained from 2-3 month old male Fischer rat adrenals depended primarily on the serum supplement, on the method of c e l l dissociation and on the f i n a l FIGURE Ll. Primary culture of normal adrenal c o r t i c a l c e l l s grown i n Waymouth*s medium, standard optics X100. a) c e l l s are mostly f i b r o b l a s t - l i k e , some with l i p i d inclusions (upper l e f t ) . b) same primary culture as i n F i g . l a , showing an area with groups of e p i t h e l i a l c e l l s (E). Table 5. The Influence of the Strai n , Age and Sex of Rat on (a) primary culture morphology and growth and (b) the a b i l i t y of explanted adrenal c e l l fragments to attain an e p i t h e l i a l morphology when grown i n secondary culture i n medium supplemented with horse serum.1 Rat Strain Sex Age (Weeks) Primary Culture Morphology2 Time of Primary Monolayer Confluence (days) Secondary Culture Morphology FISCHER male 4 mainly f i b r o b l a s t - l i k e c e l l s , 9-10 variable; from e p i t h e l i a l few l i p i d containing and epith- to f i b r o b l a s t - l i k e i a l c e l l s 7 as above 9-10 as above 10 f i b r o b l a s t - l i k e c e l l s , l i p i d 11-12 generally e p i t h e l i a l containing and e p i t h e l i a l c e l l s 12 as above 12-14 generally e p i t h e l i a l female 7 mainly f i b r o b l a s t - l i k e c e l l s , 9-10 variable; from e p i t h e l i a l few l i p i d containing and to f i b r o b l a s t - l i k e e p i t h e l i a l c e l l s 10 l i t t l e outgrowth, cultures do -not reach confluence WISTAR male 4 f i b r o b l a s t - l i k e , no l i p i d con- 9-10 f i b r o b l a s t - l i k e taining or e p i t h e l i a l c e l l s 12 i b i d 9-10 f i b r o b l a s t - l i k e Monolayer fragments were obtained from confluent primary cultures and grown on medium supplemented with horse serum. Cultures of at least 5 adrenal glands were observed for each variable. 38 c e l l density reached by the primary cultures at the time of subculture (Table 6). I f secondary cultures were grown i n medium supplemented with 10-35% FCS, they were f i b r o b l a s t - l i k e (Fig. 2a) regardless of the serum l o t , of the method of c e l l dissociation used or of the state of confluence of the primary cultures at the time of subculture. However, i f c e l l s were subcultured from confluent primary cultures mechanically, as c e l l groups, and grown i n HS-medium, then the c e l l s formed a uniform epithelium with prominent l i p i d inclusions (Fig. 2b). Cells that were t r y p t i c a l l y d i s -sociated from confluent primary cultures or subcultured from p r o l i f e r a t i n g nonstationary primary monolayers either with trypsin or mechanically, assumed shapes intermediate between a f i b r o b l a s t - l i k e and e p i t h e l i a l - l i k e form i n HS-medium but never became completely e p i t h e l i a l and did not accumulate l i p i d . The size of monolayer fragments (Table 7) cut from confluent primary cultures did not appear to influence the a b i l i t y of c e l l s to at t a i n an e p i t h e l i a l culture morphology. Fragments that were exposed to trypsin or p i p e t t i n g , although not disrupted by these treatments into single c e l l s , were f i b r o b l a s t - l i k e (Table 7). The a b i l i t y of primary monolayer fragments to develop an e p i t h e l i a l configuration upon subculture into HS-medium did not appear to be i n f l u -enced by the l o t of FCS i n which primary cultures were grown. However, the sex, age and s t r a i n of rat from which c o r t i c a l c e l l s were obtained appeared to be an important determinant for the consistent development of an e p i t h e l i a l - l i k e morphology (Table 5). Monolayer fragments obtained from c o r t i c a l explants of 4-7 week old male or female Fischer rats did not con-s i s t e n t l y develop an e p i t h e l i a l - l i k e morphology under standard culture condi-tions. Primary monolayer fragments obtained from Wistar rat adrenals, grown Table 6. Morphology of Secondary Adrenal Cultures: Influence of the Serum Supplement, Method of C e l l Dissociation and C e l l Density of Primary Cultures at Time of Subculture Serum Supplement Tryptic dissociation Mechanical dissociation Subculture from Subculture from Nonconfluent, Confluent P r o l i f e r a t i n g Cells C e l l Sheet Nonconfluent, Confluent P r o l i f e r a t i n g Cells C e l l Sheet f e t a l c a l f 10% 15% 25% 35% horse serum 2 10% f i b r o b l a s t i c f i b r o b l a s t i c f i b r o b l a s t i c f i b r o b l a s t i c f i b r o b l a s t i c f i b r o b l a s t i c f i b r o b l a s t i c f i b r o b l a s t i c intermediate intermediate f i b r o b l a s t i c f i b r o b l a s t i c f i b r o b l a s t i c f i b r o b l a s t i c f i b r o b l a s t i c f i b r o b l a s t i c f i b r o b l a s t i c f i b r o b l a s t i c intermediate e p i t h e l i a l with l i p i d inclusions The number of subcultured c e l l s per dish was i n the range of 3 to 5 x l 0 4 . Cultures of at least 10 adrenal glands were observed for each of the 20 combinations of serum, dissociation method, and c e l l confluency. Horse serum was purchased from North American Biolbgical Company, serum l o t #201189. C e l l shape and growth pattern were intermediate between a f i b r o b l a s t i c and an e p i t h e l i a l form. FIGURE 2. a) secondary culture of adrenal c e l l s , grown i n Waymouth's medium with f e t a l c a l f serum following t r y p t i c dissociation, resulting i n a f i b r o b l a s t - l i k e morphology, phase optics, X210. b) secondary culture of adrenal c e l l s grown i n Waymouth's medium with horse serum, following mechanical dissociation as c e l l groups from a confluent primary culture, resulting i n an e p i t h e l i a l - l i k e morphology with prominent l i p i d inclusions, phase optics, X210. c) primary culture of adrenal c o r t i c a l - c e l l s grown i n Waymouth's medium with f e t a l calf serum, showing a higher magnification of the e p i t h e l i a l c e l l s i n primary culture (Fig. lb) that are considered to represent highly functional fasciculata c e l l s in vitro, phase optics, X210. 41 Table 7. The Influence of the Extent of Dissociation on the A b i l i t y of Secondary Monolayers to Attain an E p i t h e l i a l Morphology.1 Type of Dissociation Size of Fragments (estimated # of c e l l s ) Morphology A. Scraping large groups e p i t h e l i a l ( > 1 0 0 c e l l s ) small groups e p i t h e l i a l ( < 1 0 0 c e l l s ) B. ( i ) single c e l l s f i b r o b l a s t - l i k e Trypsinization large groups ( > 1 0 0 c e l l s ) f i b r o b l a s t - l i k e small groups f i b r o b l a s t - l i k e ( < 1 0 0 c e l l s ) C. ( i i ) Vigorous single c e l l s f i b r o b l a s t - l i k e pipetting large groups f i b r o b l a s t - l i k e ( > 1 0 0 c e l l s ) small groups f i b r o b l a s t - l i k e . ( < 1 0 0 c e l l s ) c81cultures were~examined£for'eachctreatmenfe. 43 i n standard culture conditions, consistently yielded f i b r o b l a s t - l i k e rather than e p i t h e l i a l - l i k e monolayers. Only monolayer fragments obtained from 2-3 month old Fischer male rats appeared to consist-ently y i e l d secondary e p i t h e l i a l - l i k e c e l l s under standard culture conditions. Fibroblasts grown from muscle f a s c i a retained their fusiform shape under any of the above culture conditions. C. Tertiary and quaternary cultures a. cul_ture morpho_lo£y Secondary cultures of adrenal e p i t h e l i a l - l i k e c e l l s passaged 5 times i n HS-medium retained their symmetrical configuration. S i m i l a r l y , f i b r o b l a s t - l i k e c e l l s retained their fusiform morphology after 5 consecutive passages,.in FCS-medium. b. l ^ i f _ e ^ and_and_pr_oliferat ion The p r o l i f e r a t i v e a c t i v i t y of adrenal cultures varied with the serum supplement. Cells grew rapidly i n supplements of 10-35% FCS while i n HS-medium, the rate of c e l l growth was considerably reduced (53). In general, the stationary phase i n secondary culture was reached by f i b r o b l a s t - l i k e monolayers at a c e l l density of approximately 1 x 10 6 c e l l s / c u l t u r e dish and by e p i t h e l i a l - l i k e c e l l s at a density of about 2 x 10** c e l l s / c u l t u r e dish. The lifespan of the adrenal cultures was much shorter than that of muscle fascia fibroblasts (Fig. 3). The maximum lifespan of f i b r o b l a s t - l i k e adrenal c e l l s , with weekly passages, was 5-6 weeks, while the connective tissue fibroblasts of fascia o r i g i n were s t i l l growing rapidly after 3 months i n continuous culture. When sub-cultured once a week, the lifespan of adrenal e p i t h e l i a l - l i k e c e l l s FIGURE 3. Lifespan of adrenal epithelial-like(°-°), adrenal fibroblast-like (— >ce!ls and muscle fascia fibroblasts J 3 5 7 w e e k s i n c u l t u r e was approximately the same as that of adrenal f i b r o b l a s t - l i k e c e l l s However, u n l i k e the l a t t e r c u l t u r e s , i t appeared from the weekly c e l l counts that the e p i t h e l i a l c e l l s m u l t i p l i e d s l o w l y , i f at a l l ( F i g . 3). 46 I I . Functional Properties A. Histochemistry a. histochemical_ ^ emonstra_ti_on c^f_A5j-3_6^hyjdr_02C^^tero^id_ ^ ehyd_ro_genase_, £lucos_e-6-£_hosjoha.te^  jiehydrogenase and 3-i£id (Fig. 4) Histochemical 'reactions for A5-3g-hydroxysteroid dehydrogenase (HSD),(glucose-6-phosphate dehydrogenase (GPD) and l i p i d as demonstrated by formazah deposition and o i l red 0 respectively, were more intense i n adrenal than i n connective tissue f i b r o b l a s t s . Connective tissue f i b r o b l a s t s were essenti a l l y negative for GPD (Fig. 4h), l i p i d (Fig. 4g) but exhibited traces of formazan deposition (Fig. 4i) suggesting some SDH a c t i v i t y . Adrenal f i b r o b l a s t - l i k e c e l l s exhibited a pos-i t i v e response i n a l l three reactions (Fig. 4d-f). The intensity of staining i n adrenal e p i t h e l i a l - l i k e c e l l s (Fig. 4a-c) was greater than i n the adrenal f i b r o b l a s t - l i k e c e l l s . b. h.i£^ toch.emical ji eHionstra.tion of_acid muc^op_olys^ac^char_ides^ (Table 8) Cultures of adrenal f i b r o b l a s t - l i k e c e l l s produced ext r a c e l l u l a r matrix material that was metachromatic when stained with toluidine blue at pH 2.5 and that existed as a sheet of material closely applied to monolayers (Fig. 5b). Adrenal e p i t h e l i a l - l i k e c e l l s i n HS-medium did not produce histochemically demonstrable metachromatic extracellular matrix when stained with toluidine blue at pH 2.5 (Fig.5a). I'h). orderotoeteh'fcat&y.elyely characterize the nature of the mucin-like material produced by adrenal f i b r o b l a s t - l i k e c e l l s and determine i f charge was due to carboxyl or sulphate groups, monolayers were stained with 1. PAS/alcian blue, PH~2T5, 2. alcian blue at pH 1.0 and 2.5, 3. a l c i a n blue at pH 2.5 after methylation, saponification and hyaluronidase treatment. The positive reaction to PAS and alcian blue suggest that the charged ext r a c e l l u l a r matrix (MECM) i s car-bohydrate i n nature while lack of staining with alcian blue at pH 1.0 FIGURE 4. Histochemistry • of adrenal c o r t i c a l and muscle fascia c e l l s , standard optics, X320. a-c. cultures of e p i t h e l i a l - l i k e adrenal c e l l s stained for (a) l i p i d , (b) glucose-6-phosphate and (c) steroid dehydrogenase. d-f. cultures of f i b r o b l a s t - l i k e adrenal c e l l s stained for (a) l i p i d , (b) glucose-6-phosphate and (c) steroid dehydrogenase g - i . cultures of connective tissue fibroblasts stained for (a) l i p i d , (b) glucose-6-phosphate and (c) steroid dehyd-rogenase. Staining for both types of dehydrogenase a c t i v i t y , as w e l l as for l i p i d , i s least intensive i n connective tissue fibroblasts ( g - i ) , intermediate i n f i b r o b l a s t - l i k e adrenal c e l l s (d-f) and most intensive i n e p i t h e l i a l - l i k e adrenal c e l l s (a-c). 48 FIGURE 5. Adrenal monolayers grown i n different culture conditions, f i x e d i n 2.5% glutaraldehyde and stained with toluidine blue at pH 2.5, standard optics, X210. a. secondary cultures of adrenal c o r t i c a l epith-e l i a l - l i k e c e l l s i n HS-medium. Cells remain as cohesive groups and contain l i p i d inclusions and do not produce detectable MECM. b. secondary cultures of adrenal f i b r o b l a s t -l i k e c e l l s i n FCS-medium. Cells are bipolar, do not con-t a i n noticable l i p i d inclusions and are covered by MECM. c. secondary cultures of adrenal c o r t i c a l e p i t h e l i a l - l i k e c e l l s i n HS-medium after exposure to hyaluronic acid. Cells appear intermediate i n morphol-ogy between (a) and (b) but appear less cohesive than c e l l s i n (a). No MECM i s observed. d. secondary cultures of adrenal c o r t i c a l c e l l s grown on sulphonated surfaces i n HS-medium. Cells are f i b r o b l a s t - l i k e and do not produce observable MECM. The dark background i s due to stain uptake by negatively charged p l a s t i c surface. e. secondary cultures of adrenal c e l l s i n HS-medium grown on polylysine surfaces. Cells are f i b r o b l a s t -l i k e , widely separated from one another and do not produce detectable MECM. f. secondary cultures of adrenal c e l l s i n HS-medium grown on mechanically brushed surfaces. C e l l groups have a ragged appearance aMhough cohesive. Table 8. Staining Characteristics of Secondary Cultures of Adrenal C o r t i c a l F i b r o b l a s t - l i k e Cells Maintained i n FCS-Medium Type of Mucin A PAS/Alcian blue pH 2.5 B Alcian blue pH 1.0 C Alcian blue pH 2.5 D Methylation/Saponification Stained with Alcian blue at pH 2.5 E Hyaluronidase treated, stained with Alcian blue, pH 2.5 cartilage blue + + + e p i t h e l i a l mucin, small intestine purple + + _ + + adrenal f i b r o b l a s t - l i k e c e l l s i n FCS-medium blue-purple - + - ± -52 suggests the charge i s due to carboxyl groups. This conclusion i s sub-stantiated by lack of staining after hyaluronidase treatment and weak s t a i n -ing after saponification of methylated MECM. The a b i l i t y of hyaluronidase to degrade the MECM of adrenal f i b r o b l a s t - l i k e monolayers but not of c a r t i -lage and the a b i l i t y to stain at pH 2.5 but not 1.0 with alcian blue suggests that a major component of the matrix is.r'carboxylated and might be hyaluronic acid rather than chondroitin sulphate. Further characterization of the biochemical properties of MECM i s necessary for d e f i n i t i v e i d e n t i f i c a t i o n of i t s components. B. Steroid production a. r^^^Cjprejgnejiolon^ me^abjol_i^m_and_re_sp_onse_ to_ACTH The conversion of [ 4 - 1^^pregnenolone to metabolites with and without ACTH i s given i n Table 9. fAaithougheMt/feheiacu6ekresp6nse^ACTH does not. appear febistimu'late steroid production when pregnenolone i s used as a substrate (23), during the trophic action of ACTH, an increase i n the formation and a c t i v i t y of steroid hydroxylases that convert pregnenolone to corticosterone i s observed (23). Since cultures reported here were incubated with ACTH for a s u f f i c i e n t length of time for t h i s polypeptide to exert i t s trophic effects (73-75), an alt e r a t i o n i n pregnenolone metabolism to corticosteroid i n t e r -mediates was anticipated. Adrenal e p i t h e l i a l - l i k e c e l l s i n HS-medium not stimulated by ACTH, metabolized approximately 65% of the pregnenolone i n 8 h, producing mainly pregn-5-ene -3B,20a-diol and smaller amounts of 20ahydroxy-pregn-4-en-3-one and deoxycorticosterone. The addition of trophic hormone ddid not s i g n i f i c a n t l y a l t e r the amount of [4- 1 4]-pregnenolone metabolized by adrenal e p i t h e l i a l i l i k e cultures. However, after incubation with ACTH for 1 day, [4- 1' tC]pregnenolone was metabolized to corticosterone i n keeping with the observation that ACTH exerts a trophic effect. This response reached a maxi-mum after 3 days of preincubation with the trophic hormone (P<0.001) and appeared to drop after 5 days (P<0.005) '(Table 9A). Table 9. E f f e c t of ACTH on the Conversion of [4- 1^C] Pregnenolone to I d e n t i f i e d S t e r o i d s by S t a t i o n a r y Cultures of Adrenal E p i t h e l i a l - l i k e C e l l s (2X104 Cells/Culture)., Adrenal F i b r o b l a s t - l i k e C e l l s ( l x l O 6 C e l l s / C u l t u r e ) a n d Muscle F a s c i a F i b r o b l a s t s ( l x l O 6 C e l l s / C u l t u r e ) . C e l l Type Days of pre-i n c u b a t i o n w i t h ACTH % R a d i o a c t i v i t y 1 pregnenolone pregn-5-ene-33, 2 0 a - d i o l 20a-hydroxy-pregn-4-en-3-one deoxyco r t i c o -sterone c o r t i c o s t e r o n e A. Adrenal e p i - 0 34.95 + 1.70 27.07 + 2.89 1.51 ± 0.40 2.38 ± 0.32 0.00 t h e l i a l - l i k e 1 35.05 + 3.12 23.41 + 4.16 1.87 ± 0.46 2.43 ± 0.68 0.52 + 0.03 ( s t a t i o n a r y 3 35.14 + 3.12 23.76 + 3.52 2.0 ± 0.23 2.71 ± 0.53 1.00 + 0.08 c u l t u r e , 39.38 2.08 2x10^ c e l l s ) 5 + 19.60 + 3.13 1.04 ± 0.16 2.35 ± 0.31 0.72 + 0.05 B. Adrenal f i b r o - 0 61.24 + 3.50 8.58 + 2.50 0.00 0.00 0.00 b l a s t - l i k e 1 56.02 + 3.90 8.20 + 2.11 0.27 + 0.01 0.07 ± 0.06 0.00 ( s t a t i o n a r y 3 52.22 + 3.90 17.92 + 2.87 0.89 ± 0.59 0.12 ± 0.04 0.00 c u l t u r e , 54.51 l x l O 6 c e l l s ) 5 + 6.23 10.58 + 2.59 0.68 ± 0.25 0.23 ± 0.10 0.00 C. Adrenal f i b r o - 0 99.23 + 0.08 0.17 + 0.05 0.00 0.00 0.00 b l a s t - l i k e 1 99.13 + 0.08 0.16 + 0.04 0.005±0.002 0.001±0.001 0.00 (values i n B 3 99.04 + 0.08 0.36 + 0.06 0.018±0.012 0.002±0.001 0.00 extrapolated 99.01 0.12 0.21 0.05 to 2 x l 0 4 c e l l s 5 + + 0.014±0.005 0.005 ±0.002 0.00 D. Muscle f a s c i a 0 68.91 + 4.56 0.00 0.00 0.00 0.00 f i b r o b l a s t s 3 69.72 + 3.81 0.00 0.00 0.00 0.00 ( s t a t i o n a r y c u l t u r e , l x l O 6 c e l l s ) Means ± S.E.M. of 8 c u l t u r e s . 54 Adrenal f i b r o b l a s t - l i k e c e l l s , not stimulated by ACTH, metabolized [4- 1 1 +C]pregnenolone only to pregn-5-ene-33 ,20ct-diol. In contrast to the e p i t h e l i a l - l i k e cultures, the amount of pregnenolone converted to pregn-5-ene-33,20a-diol by the adrenal f i b r o b l a s t - l i k e cultures increased s l i g h t l y a f t e r the addition of ACTH. This response was s i g n i f i c a n t (P<0.05) after 3 days of preincubation with the trophic hormone but after 5 days, the production of pregn-5-ene-33,20c<r-diol was not s i g n i f i c a n t l y different from controls. The addition of trophic hormone to f i b r o b l a s t - l i k e adrenal cultures also stimulated the production of detectable amounts of 20-hydroxy-pregn-4-en-3-one and deoxycorticosterone (Table 9B). In order to compare the conversion of pregnenolone by adrenal cultures i n the f i b r o b l a s t - l i k e form and i n the e p i t h e l i a l - l i k e form on a per c e l l basis, the values given i n Table 9B for the stationary adrenal f i b r o b l a s t - l i k e cultures (LxlO 6 cells/culture) were extrapolated to percent conversion per 2x10^ c e l l s , i . e . the average number of c e l l s i n stationary e p i t h e l i a l - l i k e adrenal cultures (Table 9C). On the basis of 2x10^ c e l l s , the adrenal f i b r o b l a s t - l i k e c e l l s metabolized less than 1% of the added substrate compared to 65% metabolized by the adrenal c e l l s i n the e p i t h e l i a l - l i k e form. On a per c e l l basis, adrenal e p i t h e l i a l - l i k e c e l l s produced approximately 100 times more pregnen-5-ene-33,20a-diol, 1000 times more 20a-hydroxy-pregn-4-en-3-one and 200 times more deoxycorticosterone than did adrenal f i b r o b l a s t - l i k e c e l l s , even after maximal ACTH stimulation of the l a t t e r c e l l s (3 days). The presence of minute amounts of intermediate aad-renocorticocoids i n f i b r o b l a s t - l i k e cultures suggested that monolayers may be contaminated with a small population of highly functional adrenal c e l l s . Therefore the ;[;4-ltfC]pregnenolone metabolism of (1) micropopulations of adrenal c e l l s grown from 10-40 o r i g i n a l c e l l s and (2) monolayers at varying c e l l density were examined assuming that (1) a contaminating population could be diluted 55 out and (2) such a population was not l i k e l y to divide at the same rate as f i b r o b l a s t - l i k e c e l l s i n FCS-medium. Kahri (72) and Milner (75) have con-cluded that primary cultures contain only 1 highly functional adrenal c e l l type. Since these c e l l s have a very d i s t i n c t i v e c e l l morphology (Fig. 2c), "cloned" cultures of adrenal f i b r o b l a s t - l i k e c e l l s were carefully examined for the i r presence. Secondary adrenal cultures grown i n FCS-medium from 10-40 c e l l s appeared uniformly f i b r o b l a s t - l i k e and retained the a b i l i t y to produce pregn-5-ene-3g,20a-diol from . [4- 1 4C]-pregnenolone (Table 10) i n a l l of the cultures examined, suggesting that the low amounts of adrenal corticoids were produced by f i b r o b l a s t - l i k e c e l l s and not by a contaminating population of highly functional adrenal c e l l s such as described by Kahri (72). This con-clusions i s substantiated by the li n e a r relationship between pregn-5-ene-33» 20a-diol metabolism and the number of adrenal f i b r o b l a s t - l i k e c e l l s (Fig. 6). The production of [4-11+C]pregn-5-ene-33,2Qa-diol and [4- 1 1 +C]-20a-hydroxy-pregn-4-en-3-one implies the presence of an abnormally active reductive path-way (76) i n secondary cultures of. adrenal c o r t i c a l c e l l s , a phenomenon that appears to be generally true for adrenal c o r t i c a l c e l l s i n cultures (67, 77). In mouse adrenal preparations, the a c t i v i t y of 20a-hydroxysteroid:NADP o x i -doreductase i s stimulated i n the presence of NADPH. Further, i t i s known that when NAD or NADH, a cofactor s p e c i f i c a l l y required for keto-steroid dehydro-genase, i s depleted i n c o r t i c a l c e l l preparations, the reduction pathway rather than the biosynthetic pathway, i s preferred (76). The presence of these hydroxy-steroids could therefore result from i n s u f f i c i e n t c e l l u l a r NAD/NADH i n culture although the production of r e l a t i v e l y large amounts of endogenous corticosterone and the absence of endogenous 20a-hydroxylated steroids as detected by acid fluorescence argues against t h i s . The discrepancy between [4- 1 1 +C]pregnenolone studies and the endogenous fluorogenic steroid 56 FIGURE 6. Increase in pregn-5-ene-3|3,20a-diol with increasing cell number. o Q A 5 0 ~ 100 150 2 0 0 CELL NUMBER X IO 4 Table 10. [4- 1 4C] Pregnenolone 1 Metabolism by "Cloned" Adrenal C o r t i c a l F i b r o b l a s t - l i k e Cells Maintained i n FCS-Medium. Experiment I n i t i a l C e l l F i n a l C e l l % pregn-5-ene-3B, Number2 Number x 10° Number x 10 3 20a-diol 1 17 ± 4 2 ± 0.1 0.23 ± 0.09 2 34 ± 10 6 ± 0.2 0.31 ± 0.10 3 8 ± 2 4 ± 0.4 0.25 ± 0.08 Cultures were incubated with 100,000 dpm of llfC- for 8 h Each experiment represents t r i p l i c a t e samples ±S.E.M. production i s not l i k e l y to r e s u l t from the practice of extracting whole tissue i n the f i r s t instance and only media i n the second since C-20 hydroxysteroids are not intermediates (.76) and have been shown to be released into the surrounding media (670 • However, such differences are i n accord with previous observations i n in vitro systems of s t r i k i n g differences between the incorporation of exog-enous radioactive precursors and endogenous precursors into end-products (78,79 )' I t has been suggested that endogenous and exogen-ous precursors are never allowed to constitute a homogeneous pool. Possibly t h i s i s due to both the rapid turnover of the endogenous steroidseand the rate at which the exogenous pregnenolone can reach i t s proper s i t e of metabolism within the c e l l ( 7;9) which might be l i m i t e d , i n the case of cultured c e l l s , by membrane permeability, b. ^n^°J.eil0ils_P£.0°Lu^tIon of_f luorogenic_steroid The basal fluorogenic steroid production of l x l O 5 adrenal epith-e l i a l - l i k e c e l l s s . i n HS-medium, as determined by acid-induced f l u o r -excence was approximately 400 times more than that of the f i b r o b l a s t -l i k e c e l l s . The addition of ACTH to both f i b r o b l a s t - l i k e and epith-e l i a l - l i k e cellsa' resulted i n a cha r a c t e r i s t i c r i s e i n steroid prod-uction (Table 11). Endogenous production of steroid i n both morph-ologic forms increased after 1 day of preincubation with the trophic hormone, reached a maximum after 3 days and dropped after 5 days. Steroid production increased approximately 25 times i n maximally stimulated f i b r o b l a s t - l i k e c e l l s and 5 times i n maximally stimulated e p i t h e l i a l - l i k e c e l l s , a l e v e l that was approximately 70 times that of the maximally stimulated f i b r o b l a s t - l i k e c e l l s . Flubrometric analysis of the chromatographically isolated A steroids secreted by adrenal c e l l s i n either the e p i t h e l i a l or f i b -Table 11. Effect of ACTH1 on the Secretion of Endogneous Fluorogenic Steroid 2 by Adrenal E p i t h e l i a l - l i k e C e l l s , Adrenal F i b r o b l a s t - l i k e Cells and Muscle Fascia Fibroblasts, Expressed per l x l O 6 Cells/24 hr. Means ± S.E.M. of 8-10 Cultures. Days of pre- Adrenal E p i t h e l i a l -•like Cells Adrenal Fibrob l a s t - l i k e Cells Muscle Fascia Fibroblasts incubation ug fluorogenic 7 corticos- yg fluorogenic % corticos- yg fluorogenic % corticos-with ACTH steroid terone 3 steroid terone steroid terone 0 4.79 ± 0.57 65.00 0.014 ± 0.001 71.43 0.05 + 0.006 0 1 11.84 ± 0.39 0.11 ± 0.029 0.04 ± 0.003 3 24.70 ± 3.52 93.12 0.34 ±0.037 79.40 0.05 ± 0.004 0 5 5.00 ± 1.30 0.10 ± 0.031 0.04 ± 0.003 Every 24 h , culture medium i n each dish was replaced with 2 ml of fresh medium containing 200 mU of porcine ACTH. Values have been adjusted for medium blanks. Chromatographic characterization of fluorogenic steroid secreted by cultures was determined for day 0 and 3. The methylene chloride extracts of the supernatant media from 8-10 cultures of adrenal e p i t h e l i a l - l i k e c e l l s , adrenal f i b r o b l a s t - l i k e c e l l s and muscle fascia f i b r o b l a s t s , together with 0.06 yg of corticosterone-4- 1^C, 57.3 mCi/ml as an internal standard, were f i r s t chromatographed i n l i g r o i n for 24 hr on Whatman No. 1 paper impregnated with propylene g l y c o l , and then rechromatographed on S i l i c a Gel G plates i n benzene-ethyl acetate (3:1, v:v). Values were adjusted for loss of steroid during the i s o l a t i o n procedure, as assessed by loss of the radioactive standard, and expressed as a percentage for 1x10s c e l l s . r o b l a s t i c form, indicated that corticosterone was the major end-product released into the surrounding medium. No fluorescence was detected i n the chromatographic areas of [4- 1^c]pregnenolone, [ 4- 1 L fC]-progesterone, ^ - ^ ^ d e o x y c o r t i c o s t e r o n e or [1,2-§fl(N) ]20a-hydroxy-pregn-4-en-3-one, although the l a s t has been shown to fluoresce weakly (67). In order to ascertain whether adrenal c o r t i c a l c e l l s were able to respond to other trophic hormones and not s p e c i f i c a l l y to ACTH, the response to LH, a protein hormone capable of stimulating steroid production of the embryologically closely related ovary (sb''-.') w a s examined. Adrenal c o r t i c a l f i b r o b l a s t - l i k e c e l l s did not respond steroidogenically or morphologically to LH (Table 12) and LH had no effect on MECM production or on c e l l d i v i s i o n . c' mor_pbj3l.ojgic ch^ng_e^,_a]_te_ra.tion ln_ME_CM j^ r'£du_ct1ion_an_d_inc_rea^ se_ A n_ ci!Ai j l i v i ^ i c ^ n _ i n jrespcjise_to ACTH The trophic steroidogenic response to ACTH was accompanied by morphologic changes; alterations i n MECM production and c e l l d i v i s i o n . Without ACTH treatment, c e l l s i n the e p i t h e l i a l form were flattened, cohesive and contained numerous l i p i d inclusions (Fig. 7a). After 1 day of ACTH treatment, the c e l l s had retracted and fewer l i p i d i n -clusions were observed (Fig. 7b). By 3 days of ACTH treatment, i.e. at the time of the maximal steroidogenic response to the trophic hormone, most l i p i d inclusions had disappeared and c e l l s appeared more symmetrical and rounded than i n the controls (Fig. 7c). Cells treated for 5 days with ACTH appeared essent i a l l y similar to those treated for 3 days, though edges of c e l l monolayers tended to draw away from the substratum (Fig. 7d); many c e l l groups had l i f t e d from the culture surface e n t i r e l y . C e l l d i v i s i o n was not stimulated by 61 Table 12. The Effect of LH on Steroid Production, C e l l D i v i s i o n and Ext r a c e l l u l a r Matrix Production of Adrenal C o r t i c a l F i b r o b l a s t - l i k e C e l l s i n FCS-medium. Treatment Steroid Production Ug/24/h /10 6 C e l l s Presence of Metachromasia Detected by Toluidine Blue Mit o t i c Index per 1000 c e l l s controls <0.005 :H-+ 1.5 + 0.9 ACTH-treated (3 days) 0.15 + 0.04 + 6.3 ± 1.8 LH-treated (3 days) <0.005 trace +++ 1.4 ± 0.1 lOOmU/ml medium of l u t e i n i z i n g hormone (LH) (Bovine, NTH, Bethesda, Md) were added d a i l y to cultures of confluent adrenal f i b r o b l a s t -l i k e c e l l s together with fresh FCS-medium. FIGURE 7. The influence of ACTH on the c e l l u l a r morphology of adrenal e p i t h e l i a l - l i k e c e l l s , phase optics, X200. a. monolayer grown without ACTH. Cells are flattened, cohesive and contain prominent l i p i d inclusions. b. monolayer treated for 1 day with 200 mU ACTH. Cells have retracted and l i p i d inclusions are less numerous. c. monolayer treated for ;3 days with 200mU ACTH. Cells are rounded, symmetrical and l i p i d inclusions have disappeared. Fluorogenic steroid production has reached a maximum. d. monolayer treated for 5 days with ACTH. Cells are morphologically similar to c but entire monolayer i s starting to l i f t from the substratum. FIGURE 8. The influence of ACTH on the c e l l u l a r morphology of adrenal f i b r o b l a s t - l i k e c e l l s , phase optics, X200. a. monolayer grown without ACTH. Cells are bipolar. b. monolayer treated for 1 day with ACTH. Ce l l s are morphologically similar to a. c. monolayer treated for 3 days with 200mU ACTH. Cells are more e p i t h e l i a l - l i k e . Fluorogenic steroid production has reached maximum. d. monolayer treated -for 5 days with 200mU ACTH. Cells are morphologically similar to c. ACTH when cultures were grown i n HS-medium (Table 13D). The culture morphology of f i b r o b l a s t - l i k e c e l l s was s i m i l a r to controls a f t e r one day of ACTH treatment (Fig. 8a,b). By 3 days of trophic hormone treatment, i e . at the time of maximal s t e r o i d -ogenic response, c e l l s were more rounded and e p i t h e l i a l - l i k e and con-tained l i t t l e MECM i n comparison to controls and 1 day cultures. This morphology and lack of MECM remained e s s e n t i a l l y unchanged a f t e r 5 days of ACTH treatment (Fig. 8c,d). C e l l d i v i s i o n , as assessed by the number of m i t o t i c figures per 1000 c e l l s , was s i g n i f i c a n t l y greater (P<0.001) i n 3 and 5 day treated cultures than i n controls and 1 day treated cultures (Table 13A). Adrenal f i b r o b l a s t - l i k e cultures often (approximately 40% of the time) did not respond morphologically to ACTH even a f t e r 5 days of pretreatment with this hormone. In these cases, s t e r o i d production increased to that of cultures treated with ACTH for one day only and cultures i n v a r i a b l y retained large amounts of MECM (Table 14). C e l l d i v i s i o n was not stimulated i n these cultures even a f t e r 5 days of exposure to ACTH (Table 13B) suggesting a n c i n v e r s e i c o r r e l a t i o n betweentmitot-iw i n FCS-medium. If secondary cultures of adrenal f i b r o b l a s t - l i k e c e l l s were o r i g i n a l l y grown on FCS-medium but transferred to HS—medium during treatment with ACTH, morphological changes occurred and matrix .-disappearedvbut;.ari,increase i n m i t o t i c figures was not observed (Table 13) suggesting that factor(s) present i n FCS are required to mediate a mitogenic e f f e c t of ACTH. Table 13. . The Influence of ACTH on Adrenal C o r t i c a l C e l l Division i n the Presence of FCS-medium or HS-medium. Original Culture Morphology Serum Supplement Days of ACTH pretreatment Culture Morphology After Treatment \ " With ACTH Mito t i c Index 3 per 1000 Cells A. f i b r o b l a s t - l i k e FCS-medium 0 f i b r o b l a s t - l i k e 4.1 ± 0.85 1 f i b r o b l a s t - l i k e 3.2 ± 0.63 3 e p i t h e l i a l - l i k e 14.2 ± 1.85 5 e p i t h e l i a l - l i k e 13.6 ± 1.51 B. f i b r o b l a s t - l i k e 1 FCS-medium 3 f i b r o b l a s t - l i k e 3.5 ± 0.41 5 f i b r o b l a s t - l i k e 4.8 ± 0.10 C. f i b r o b l a s t - l i k e 2 FCS-medium trans- 0 f i b r o b l a s t - l i k e <1 ferred to HS-medium 1 f i b r o b l a s t - l i k e <1 upon treatment with 3 e p i t h e l i a l - l i k e '. <1 ACTH 5 e p i t h e l i a l - l i k e <1 D. e p i t h e l i a l - l i k e HS-medium 0 e p i t h e l i a l - l i k e <1 1 e p i t h e l i a l - l i k e <1 3 e p i t h e l i a l - l i k e <1 5 e p i t h e l i a l - l i k e <1 Adrenal f i b r o b l a s t - l i k e cultures that did not respond morphologically to ACTH although steroid production increased. Monolayers were o r i g i n a l l y grown i n FCS-medium but were changed to HS-medium pr i o r to and during ACTH treatment. Means ± S.E.M. of 6 cultures. Table 14. The Relationship Among Secondary Adrenal Culture Morphology, Presence of MECM and Endogenous Steroid Production of Cells Maintained i n FCS-medium i n the Presence of ACTH. Days of Pretreatment Culture Morphology Steroid Production2 Presence of MECM With ACTH ug/106 c e l l s /24h Metachromasia Bound Dye 1 , z 0 f i b r o b l a s t - l i k e 0.01 ± 0.005 ++ 0.14 ± 0.01 1 f i b r o b l a s t - l i k e 0.12 ± 0.04 ++ 0.09 ± 0.06 3 e p i t h e l i a l - l i k e f i b r o b l a s t - l i k e 0.44 ± 0.109± 0.10 0.022 + ++ 0.06 ± 0.006 0.08 ± 0.005 After e q u i l i b r a t i o n with toluidine blue at pH 2.5, bound dye was eluted with acid methanol and measured c o l o r i m e t r i c a l l y . Values represent means ± S.E.M. of 8 samples. 66 The ACTH-induced stimulation of adrenal c o r t i c a l c e l l d i v i s i o n i s in t e r e s t i n g i n view of the considerable controversy regarding the mito-genic properties of t h i s hormone. In vivo studies suggest that ACTH i s responsible for hyperplasia of the adrenal cortex although whether t h i s action i s d i r e c t or i n d i r e c t i s not known (23). In vitro studies have provided c o n f l i c t i n g r e s u l t s with some investigators reporting stimul-ation of c e l l d i v i s i o n (81,82) and ethers reporting an i n h i b i t i o n of c e l l d i v i s i o n (83). The r e s u l t s reported here ind i c a t e that ACTH by i t s e l f i s not mitogenic since addition of t h i s hormone to stationary c e l l s main-tained i n HS-medium d i d not promote c e l l d i v i s i o n . I t appears that a fac-tor present i n FCS i s required to mediate the e f f e c t of ACTH. Whether ACTH "primes" the cells,which then require a subsequent mitogenic protein ( i e . i n s u l i n ) . t o complete t h i s e f f e c t ; o r whether components i n FCS met-abolize ACTH to an a c t i v e l y mitogenic hormone i n a manner s i m i l a r to the production of somatomedin (84) i s not clea r . The co n s i s t e n t . c o r r e l a t i o n between increased m i t o t i c a c t i v i t y , presence of ACTH and disappearance of MECM suggests that ACTH may exert, at le a s t part,of i t s e f f e c t on c e l l m itosis by degrading MECM,,which i n turn.*vmay"all6w>"a surge of c e l l d i v i s i o n i n the presence of mitogenic factors found i n FCS-medium. However, i t i s i n t e r e s t i n g that mitosis was stimulated when there was a t r a n s i t i o n between e i t h e r of the two c e l l types, i n i t i a t e d by ACTH upon modulation of a f i b r o b l a s t - l i k e form to an e p i t h e l i a l - l i k e form or by the add-itions- of FCS-medium causing an a l t e r a t i o n of an e p i t h e l i a l - l i k e morphology to a f i b r o b l a s t - l i k e morphology. Since i n the f i r s t case, there was a loss of MECM yet i n the second case MECM production was stimulated, i t i s un l i k e l y that thehmeGhaniis*miid.f.GmitotrGms.fimuliati'on wasiisimilarbin-both, cases. 67. d. [iL--.^]p_re_gneno].one_me_t^bo_li^sm,_en_djDgenous_ JTu^L°&eB.i£L s^ te_ro^ id_ p_ro^duct_ioji_and_re_s2.onse_ ^ .o_ACTH o.f_c^nt_rc^l_c^lt_ure_s c^f_mus^le_ f_as_cia f_ib_robl_as_ts^. Muscle fascia fibroblasts metabolized approximately the same amount of [4- 1 1 +C]pregnenolone as the adrenal f i b r o b l a s t - l i k e c e l l s , but converted i t to unidentified polar compounds only and did not produce pregn-5-ene-33,20a-diol, 20a-hydroxypregn-4-en-3-one or deoxycorticosterone. They also did not respond to ACTH (Table 9D). Although cultures of muscle fascia fibroblasts did produce f l u o r -ogenic material (Table 11), the monolayers did not respond-to ACTH or produce corticosterone. Culture morphology of muscle f a s c i a f i b -roblasts did.not change i n response to ACTH when c e l l s were grown i n HS-medium or i n FCS-medium. I I I . Ultrastructure of adrenal c o r t i c a l c e l l s In HS-medium and i n  FCS-medium with and without ACTH A. Adrenal e p i t h e l i a l - l i k e c e l l s i n HS-medium a. without^ACTH Toluidine blue staining of monolayers of adrenal e p i t h e l i a l - l i k e c e l l s distinguished two c e l l types: darkly staining c e l l s located at the periphery of c e l l groups and l i g h t l y staining c e l l s located toward the centre of a c e l l group. Ce l l u l a r uptake of o i l red 0 suggested that the l i g h t appearance was due to an abundance of l i p i d . U l t r a s t r u c t u r a l l y , c e l l s at the centre of a group could be d i s t -inguished from those at the periphery, i ) inner c e l l s Cells towards the centre of a c e l l group contained large i n c l -suions of electron lucent material shown to be l i p i d on the basis of o i l red 0 uptake and often exhibiting a c r y s t a l l i n e structure (Fig. 9a). These l i p i d containing c e l l s generally comprised the majority of c e l l s within a group. Cell s contained numerous mitoch-ondria with c l a s s i c a l UamfiiLlFar cristae although some mitochondria contained a dense matrix with only a few c r i s t a e (Fig. 9a). Cells contained small amounts of smooth endoplasmic reticulum (SER) and only an occasional Golgi apparatus (Fig. 9a). ; Poorly developed rough endoplasmic reticulum (RER) was more common than SER. Loose ribosomes abounded i n the cytoplasm. Plasma membranes were closely apposed to one another forming extensive intermediate junctions sep-arated by a 200-300 % gap and interrupted by focal widening between membranes to-form what appear to be small channels between c e l l s . Numerous pinocytotic wes-rcTess were observed at the surface mem-brane (Fig. 9a). Few m i c r o v i l l i appeared on the c e l l surface. No FIGURE 9. Ultrastructure of adrenal c o r t i c a l e p i t h e l i a l - l i k e c e l l s i n HS-medium without ACTH. a. inner l i p i d containing c e l l s : large cryst-a l l i n e l i p i d inclusions (L) are abundant, rough endoplasmic reticulum (re) present, numerous mitochondria with lamellar cris t a e and occasional Golgi (g) are observed. Cells are closely adherent to one another and pinocytotic vessicles (p) are seen at the c e l l surface, X13,800. b. c e l l s appear to be attached to long c e l l processes rather than d i r e c t l y to the substratum (arrow indicates substratum), X22,540. c. outer c e l l s contain less l i p i d than inner c e l l s and that present i s amorphous rather than c r y s t a l l i n e . Large amounts of Golgi (g) and smooth endoplasmic reticulum (se) are observed. Mitochondria have lamellar c r i s t a e , X 13,800. d. gap-like junctions- occurring as c i r c u l a r inclusions within outer c e l l cytoplasm, X27,540. 70 other specialized junctions between c e l l s were observed either at the substrate or medium edge of a c e l l colony. No e x t r a c e l l u l a r material was observed between c e l l s . Microtubules and microfilaments were rarely observed. Sections cut perpendicular to the growth surface showed that the majority of c e l l s did not attach d i r e c t l y to the substratum but rather.to elongated c e l l processes extending p a r a l l e l to the growth surface (Fig. 9b). i i ) outer c e l l s Cells did not contain large c r y s t a l l i n e l i p i d inclusions but exhibited small inclusions of highly electron dense amorphous l i p i d . Mitochondria were numerous and contained laminar cristae. SER was abun-dant and dilat e d Golgi saccules were common (Fig. 9c). RER was almost e n t i r e l y absent, and ribosomes occurred as polysomes rather than as free e n t i t i e s . Whorled membranes (Fig. 9c) were common and lysosomes abun-dant. Penti'laminar gap-like junctions as w e l l as gap junctions exhib-i t i n g a 20& gap were observed within the cytoplasm (Fig. 9d). C e l l surfaces exhibited many m i c r o v i l l i , b. one d.ay_ of_ACTH treatmen_t i ) inner c e l l s Inner c e l l s after 1 day of exposure to ACTH showed an increase i n polysome formation. Ribosomes on RER were widely spaced and fewer than i n controls (Fig. 10b). SER and Golgi occurred i n approximately the same number as controls. L i p i d inclusions appeared less dense (Fig. 10a). Cells had broken apart from one another and appeared attached over small areas only (Fig. 10a). No microfilaments were observed at the areas of detachment (Fig. 10b). Pinocytotic vesi'c'less were less numerous than i n controls and i n increase i n surface m i c r o v i l l i was observed (Fig. 10a). FIGURE 10. Ultrastructure of adrenal c o r t i c a l e p i t h e l i a l -l i k e c e l l s treated with 200mU ACTH for 1 day. a. inner c e l l s : polysome formation has increased, l i p i d inclusions appear less dense although s t i l l c r y s t a l l i n e , mitochondria r e t a i n l a m i l l a r c r i s t a e stueture. Cel l s have retracted from one another, X.vl3?,800. b. higher magnification of (a): rough endoplasmic reticulum (arrow) contains fewer ribosomes than controls. No microfilaments are see along areas of c e l l detachment, X 46,000. c. outer c e l l s have rounded up and show increased m i c r o v i l l i (m) at the c e l l surface. There i s an increase i n smooth endoplasmic reticulum (se) and Golgi (g) as well as i n the number of whorled bodies (arrow). Cel l s appear to be detaching from c e l l processes (cp) next to the substratum, X 13,80,0;. d. higher magnification of whorled bodies, X 22,000. e. higher magnification of microfilaments present i n c e l l process next to the substratum (cp) but not i n the detaching c e l l body, X 46,000. f. gap-like junctions appear at the ends of retracting c e l l processes, X 8,000. g. gap-like junction i n (f) magnified: the occasional 20 %. gap can be observed (arrow), X 46,000. 73 74 i i ) outer c e l l s Cells at the periphery had rounded up (Fig. 10c) but c e l l - c e l l contact persisted v i a long processes (Fig. l O f ) . The rounding and presence of long c e l l processes corresponded to the s t e l l a t e appear-ance of peripheral c e l l s after one day of ACTH treatment, observed at the l i g h t microscope l e v e l . At the t i p s of adjoining c e l l proc-esses, pentilaminar junctions were often observed (Fig. lOf,g) that sometimes appeared gap-like i n structure (Fig. lOg). Many c e l l s were s t i l l adherent to the long c e l l processes that r^n p a r a l l e l to the sub-stratum observed i n control cultures. However, the extent of attach-ment to these processes had decreased (Fig. 1 0 c ) . Large bundles of microfilaments were observed within the underlying processes (Fig.lOe) but not within the rounding c e l l s (Fig. lOe). An increase i n polysome formation and a large increase i n the number and d i l a t i o n of SER as well as Golgi saccules were observed but the number and morphology of the mitochondria remained similar to controls (Fig. 1 0 c ) . An increase i n whorled membranous bodies was observed (Fig. lOd) and numerous m i c r o v i l l i occurred on the c e l l surface (Fig. 1 0 c ) . The appearance of gap-like junctions after stimulation with ACTH for 1 day has hot been described before i n the adrenal cortex in vivo or in vitro although G i l u l a and Friend 085"0 have i d e n t i f i e d the presence of gap junctions between adrenal c e l l s i n unstressed animals. Extensive gap junction formation occurs i n many " d i f f e r e n t i a t i n g " tissues (86^88?-) and jfeass been shown to increase between granulosa c e l l s i n response to hormonal stimulation as f o l l i c l e s mature 089,90 )• Similar to the case reported here, the formation of these junctions corresponded to the development of steroid responsiveness to p i t u i t a r y hormones ('89*) • The gap, as w e l l as tight junction, has been implicated as the s i t e of metabolic coupling (91 ) between c e l l s and i n t h i s cap-a c i t y , junction formation may ensure coordinated response to hormonal or morphogenetic s t i m u l i (90 .). Many authors (92 ) have shown that large molecules can be transmitted between c e l l s suggesting that c y c l i c nucleotides might also be passed from c e l l to c e l l . Prelim-inary work by Sheridan (:93) indicates that t h i s i s the case for some c e l l l i n e s . However t h i s phenomenon has not been demonstrated for endocrine tissues as yet. Passage of c y c l i c nucleotides i s of potential importance i n tissues such as the adrenal cortex to ensure a maximal response of '.the entire gland to stress p a r t i c u l a r l y since trophic hormone receptors and the presence of ACTH-stimulated adenyl cyclase enzymes appear to be unevenly distributed within the cortex (89V ,90)). Gap junctions and t i g h t junctions have also been implicated as s i t e s of c e l l adhesion (188?) and several investigators (l'94-,95-') have suggested that these junctions help to secure the retracting granulosa c e l l s to one another during f o l l i c u l a r maturation. The predominance of pentilaminar junctions at the end of e p i t h e l i a l - l i k e cell'" processes suggests an adhesive function i n the adrenal c o r t i c a l c e l l s as w e l l . D e f i n i t i v e i d e n t i f i c a t i o n of gap as compared to tight junctions rests upon 4 c r i t e r i a (91°. 96^ 9&i->~)' which include: 1. a width of approximately 180 ft or one that i s greater than the width of two unit membranes; 2. the presence of a 20 ft gap between the two apposed outer membrane l e a f l e t s ; 3. the.penetration by lanthanum of t h i s 20 A gap; and 4. the appearance i n freeze fracture preparations of spec-i f i c arrangements of membrane p a r t i c l e s . The appearance of gap junctions i n u l t r a t h i n section preparations has been shown to vary 76 with the method used f or t i s s u e f i x a t i o n as w e l l as the sequence of stai n i n g C99..100.') • Thus, generally a 20 £ i s not present i n tis s u e dehydr.ated' p r i o r to en bloc uranyl acetate staining while best preparations were observed when c e l l s were stained en bloc with uranyl acetate p r i o r to dehydration. : C e l l s examined here were dehydrated p r i o r to staining with uranyl acetate which may explain the i n a b i l i t y to demonstrate a 20 X gap always. However, the width of the junctions was suggestive of gap rather than t i g h t junctions although c r i t i c a l i d e n t i f i c a t i o n w i l l require further i n v e s t i g a t i o n , c. 3 days of ACTH treatment At the l i g h t microscope l e v e l a f t e r 3 days of exposure to ACTH, i t was not possible to c l a s s i f y c e l l s on the basis of l i p i d i n c l u s i o n s since these had e n t i r e l y disappeared (Fig. 11a). U l t r a s t r u c t u r a l l y , considerable c e l l necrosis and degenerating'^+£ells£wer%< seen./p= •' and i d e n t i f i e d by the fragmented nuclear material and ground cytoplasm (Fig. l i e ) . Mitochondria i n these degenerating c e l l s appeared swollen so that c r i s t a e looked almost -vve'slttttaas: while RER was d i l a t e d and almost e n t i r e l y f r ee of ribosomes. L i p i d inclusions from degenerated c e l l s appeared to have been released into the medium (Fig. 12b). The remaining c e l l s were rounded, did not exhibit long c e l l processes as i n day 1 treated c e l l s , showed marked increases i n SER and Golgi saccules (Fig. 11a,c). RER also was d i l a t e d and contained granular material (Fig. l i e ) . Polysomes appeared to have increased i n number and length (Fig. 11a). L i p i d i n c l u s i o n s that remained were small, dark and amor-phous, completely lacking a c r y s t a l l i n e structure. Inclusions i n these c e l l s could be seen i n the process of being released into the surr-ounding medium (Fig. 12a) and apparently empty 'va'cuoless were commonly observed, presumably having released l i p i d contents. Large amounts of FIGURE 11. Ultrastructure of adrenal c o r t i c a l e p i t h e l i a l -l i k e c e l l s treated with 200mU ACTH for 3 days. a. c e l l s no longer contain c r y s t a l l i n e l i p i d but amorphous liposomes. Lysosomes are apparent, mitochondria s t i l l exhibit lamellar c r i s t a e , Golgi (g) and smooth endo-plasmic reticulum (s) have increased. Polysomes are s t i l l apparent, X 13,800. b. m i c r o v i l l i at the c e l l surface, X 13,800. c. microfilaments occur perpelndicular to the c e l l surface, X 13,800. d. same as (c) but higher magnification, X 46,000. e. necrotic c e l l s (dc) are common. C e l l contact observed i n Fig. 9b i s disrupted. Rough endoplasmic r e t i c -ulum i s di l a t e d , X 13,800. FIGURE 12. Ultrastructure of adrenal c o r t i c a l e p i t h e l i a l -l i k e c e l l s treated with 200mU ACTH for 3 days (a-b) and for 5 days (c-d). a. l i p i d droplet i n process of being released from healthy c e l l , X 12,000. b. l i p i d droplets being released by dying c e l l s , X 12,000. c. adrenal c e l l s treated with ACTH for 5 days. Mitochondria appear condensed but lamellar cristae are s t i l l observed. Smooth endoplasmic reticulum (s) and Golgi (g) have increased, X 15,000. d. same as (c) but containing l i p i d droplets which are electron dense and amorphous, X 15,000. 80 microfilaments occurred next to the surface membrane and i n some cases appeared to penetrate the membrane and extrude into the surr-ounding medium (Fig. 11c,d). In f a c t , masses of filaments occurred loose between c e l l s and may have been released by degenerating c e l l s . Microtubules were distr i b u t e d throughout the cytoplasm (Fig. l i d ) . C ells appeared to have separated from one another more extensively than previously and were juxtaposed only over small areas of unspec-i a l i z e d intermediate junctions. Pentilaminar junctions were not observed. Numerous m i c r o v i l l i were observed at the c e l l surface (Fig. l i b ) . Upon stimulation with ACTH, l i p i d released from the c e l l s l o c -ated toward the centre of a group could provide ready-made steroid precursors for the synthesis of adrenocorticoids by the remaining c e l l s , as has been postulated to occur in vivo ((&'6)l) . The l y p o l y t i c effect of ACTH on adrenal e p i t h e l i a l - l i k e c e l l s i s interesting i n that at least two mechanisms of l i p i d release can be observed, includ-ing c e l l necrosis and release of entire l i p i d inclusions without app-arant rupture of the entire c e l l . The l a t t e r mechanism has been described i n d e t a i l by Rhodin (QiQ'ili) and thought by him to represent a means for release of stored adrenocorticoids, although more recent data indicate that only cholesterol i s stored i n liposomes ClQ2). The widespread necrosis of l i p i d containing c e l l s observed i n the centre of c e l l groups i n the present study i s similar to that observed but not often reported, i n the zona fascicular c e l l s (103) and thought to represent either an exaggerated response to ACTH ("overwork") or the consequence of extensive membrane rupture upon attempt to release simultaneously a large number of l i p i d inclusions. d. 5 days of ACTH treatment After 5 days of ACTH treatment, many c e l l sheets had retracted from the substratum e n t i r e l y and c e l l s generally had a rounded morph-ology. U l t r a s t r u c t u r a l l y , SER was abundant, numerous apparently empty vacuoless were present, possibly representing released liposomes or RER that had l o s t ribosomes and jgr-anuitfarc matrix. Remaining RER had fewer attached ribosomes and less dense matrix than c e l l s treated for 3 days with ACTH. I n t r a c e l l u l a r microfilaments had disappeared although filamentous material could s t i l l be observed e x t r a c e l l u l a r l y . Polysomes were infrequent and ribosomes existed as single e n t i t i e s i n the cytoplasm (Fig. 12c). L i p i d inclusions were infrequent and were small and amorphous (Fig.l2d). B. Adrenal f i b r o b l a s t - l i k e c e l l s i n FCS-medium a. without ACTH Adrenal f i b r o b l a s t - l i k e c e l l s grown without ACTH and viewed at the l i g h t microscope l e v e l were bipolar and produced copious amounts of metachromatic ext r a c e l l u l a r matrix that appeared to layer c e l l s . At the u l t r a s t r u c t u r a l l e v e l , c e l l s appeared widely separated from one another except at small points of attachment (Fig. 13a). The wide extr a c e l l u l a r space between c e l l s contained filamentous material dotted with electron dense p a r t i c l e s . A similar material was often seen to be closely apposed to the c e l l surface where i t resembled basement menbrane (Fig. 13 a). This basement membrane-like material appeared to have structural i n t e g r i t y i n that i t appeared as i f i t were pulled taut beneath the c e l l surface (Fig. 13b). Immediately beneath the c e l l membrane, a large band of microfilaments was l o c a l i z e d (Fig. 13d). Extensive bands of tonofilament-like structures were ob-served to criss-cross within the inner cytoplasm (Fig. 13c). Few FIGURE 13. Ultrastructure of adrenal c o r t i c a l f i b r o b l a s t -l i k e c e l l s i n FCS-medium grown i n the absence of ACTH. a. c e l l s are coated with basement membrane (arrow). Dilated rough endoplasmic reticulum i s a prom-inent organelle. Mitochondria have lamellar c r i s t a e , X 13,000. b. basement membrane (arrow) appears to have structural i n t e g r i t y . X 22,540. c. large tonofilament-like structures are evident, occasional droplets of l i p i d are seen. Rough endoplasmic reticulum i s d i l a t e d and f i l l e d with granu'larr material, X 28,750. d. microfilaments and microtubules (mt) are aligned p a r a l l e l to the membrane, X 22,540. 84 mitochondria were observed but could be c l a s s i f i e d as two types; large elongate mitochondria with lamellar cristae and small round mitochondria with dense matrix and few cris t a e (Fig. 13d). Dilated RER was ubiquitous and the most prominent organelle. A few free and polysomal ribosomes were also observed. Occasional amorphous liposomes were present. Very l i t t l e SER and few Golgi were observ-ed although occasional c e l l s contained considerable quantities of both (Fig. 13a). b. 1 day_ £f_ACTH t r ea± ment Adrenal f i b r o b l a s t - l i k e c e l l s treated for one day with ACTH, morphologically resembled control cultures at the l i g h t microscope level.. U l t r a s t r u c t u r a l l y , c e l l s were s t i l l generally widely separated from one another although there was indication of increasing c e l l -c e l l contact (Fig. 14a): extracellular spaces appear narrower and base-ment membrane was not as extensive as i n controls. Microfilaments and microtubules were s t i l l present i n abundant quantities (Fig. 14 c, d). Dilated RER remained a prominent organelle. An increase i n pentilaminar junctions was observed between c e l l s (Fig. 14a,b). c. _3 d_ay_s_of_ ACT_H_tr_ea_tment Adrenal f i b r o b l a s t - l i k e c e l l s appeared rounded i n configuration or e p i t h e l i a l - l i k e at the l i g h t microscope:Me.vel and MECM had d i s -appeared. U l t r a s t r u c t u r a l l y , the ext r a c e l l u l a r space was reduced (Fig. 15c) and c e l l s had approached one another so that extensive areas of apposed membranes were observed. Basement membrane-like material had disappeared (Fig. 15c). An increase i n SER and Golgi were observed, RER had collapsed (Fig. 15d) and polysomes as well as single ribosomes abounded i n the cytoplasm (Fig. 15c). The extensive bundles of microfilaments observed i n the cytoplasm of FIGURE 14. Ultrastructure of adrenal c o r t i c a l f i b r o b l a s t -l i k e c e l l s i n FCS-medium treated for 1 day with 200mU ACTH. a. c e l l s are essenti a l l y similar to controls (Fig. 13) although less basement membrane i s apparent and c e l l - c e l l contact i s more extensive (large arrow). Gap junctions are numerous (small arrow), X 13,000. b. gap junction i n (a) magnified. Most of the junction appears pentilaminar although i n places a 20 ft gap can be'. seen (arrows), X 46,000. c. large microfilaments occur within the cyto-plasm. Mitochondria exhibit lamellar c r i s t a e , X 28,800. d. microfilaments aligned p a r a l l e l to the c e l l surface,'X 22,500. FIGURE 15. Ultrastructure of adrenal c o r t i c a l f i b r o b l a s t -l i k e c e l l s i n FCS-medium treated with 200mU ACTH for 3 days (a-d) and for 5 days (e). a. c e l l s exposed to ACTH for 3 days: m i c r o f i l -aments are aligned perpendicular to the c e l l surface (arrow) and basement membrane ,material has disappeared, X 13,800. b. c e l l s exposed to ACTH for 3 days: same as i n (a) but higher magnification showing alignment of micro-filaments, X 46,000. c. c e l l s treated with ACTH for 3 days: basement membrane has disappeared and mitochondria exhibit l a m i l l a r c r i s t a e , X 13,800. d. same as (c), Golgi (g) and smooth endoplasmic reticulum (r) have increased, X 13,000. e. c e l l s treated with ACTH for 5 days. The number of mitochondria have increased but s t i l l exhibit . lamellar c r i s t a e , Golgi (g) and smooth endoplasmic r e t i c -ulum (r) have increased, X 22,400. 90 c e l l s treated for one day with ACTH, were reduced and i n many cases appeared to have disappeared (Fig. 15d). However, extensive arrays of microfilaments remained next to the c e l l surface running perpen-dicular to the membrane rather than p a r a l l e l as observed previously (Fig. 15a,b). Mitochondria did not appear to have increased i n number and cristae remained laminar, d. f_ive_days_ c^f_ACTH t^ re_atmen_t Adrenal f i b r o b l a s t - l i k e c e l l s treated for 5 days resembled c e l l s treated for 3 days with ACTH at the l i g h t microscope l e v e l . U l t r a -s t r u c t u r a l l y , c e l l s also resembled those treated for 3 days with ACTH. Some debris, presumably due to c e l l necrosis was observed, SER and the number of mitochondria appeared to have increased. There were no changes i n mitochondrial cristae (Fig. 15e). 91 IV. The r o l e of serum supplements, growth surfaces and subculture  techniques i n determining culture morphology, c e l l movement, MECM  production and s t e r o i d secretion. A. Serum supplements a. rever_sjLbi.li_t_y_ o^ f_se_c£nd_ar3^ _cu_l^ ur_e_mo^ rp_ho^ logy_ ^on_sub^eaue_n_t j3ub_cult_ure_ As outlined i n . TablecrlS', adrenal c o r t i c a l monolayers were subcultured sequentially from FCS-medium to HS-medium and from HS-medium to FCS-medium i n order to assess the a b i l i t y of secondary cultures of f i b r o b l a s t - l i k e c e l l s maintained i n FCS-medium or ep-i t h e l i a l - l i k e c e l l s maintained i n HS-medium to revert to an ep i t h -e l i a l - l i k e or f i b r o b l a s t - l i k e form r e s p e c t i v e l y upon subculture into appropriate culture conditions. As indicated i n Figure 16, c e l l s that had been maintained as f i b r o b l a s t - l i k e ( i e . i n FCS-medium) fo r up to 3 subcultures retained the a b i l i t y to revert to an ep i t h -e l i a l - l i k e form (Fig. 16d,e) i f subcultured as monolayer fragments and grown i i i HS-mediuffii /Such c e l l s seemedbableota G0r£i'costter,oneeande[4-^ [. Aek^c ] p regneno-lone^asPinddtGatednby chebmatographyVe Fi>i3r6blras.fer-tLitketm6noil!ay.ers' maintained-. iindFGS-mediumsfore4tse4uenti"altsubculturestditdtnot - a t t a l n - t a n l e p i t h e l i a l -l i k e form upon subsequent subculture into HS-mediumi) (Fig. 16f) and did not produce [4- 1 1 +C]deoxycorticosterone and [4- 1^C]20a-hydroxy-pregn-4-en-3-one. E p i t h e l i a l - l i k e c e l l s maintained i n HS-medium retained the a b i l i t y to assume, upon subculture into FCS-medium, a f i b r o b l a s t - l i k e morphology even a f t e r 4 sequential subcultures, '(Fig. 16a-c). This morphologic change was accompanied by a s h i f t to the steroidogenic pattern c h a r a c t e r i s t i c of f i b r o b l a s t - l i k e c e l l s / Table 15 Outline of Experiment Designed to Assess the Continued A b i l i t y of Adrenal C o r t i c a l Monolayers to Al t e r Morphology and Functional Expression When Grown In Medium Supplemented with FCS or Medium Supplemented with HS. F i r s t Second Third Fourth FCS- FCS HS* FCS Primary MONOLAYERS [4- 1 1 +C] pregnenolone metabolism assay: 20a,-hydroxypregn-4-en-3-one .-(-)• = presence of deoxycorticosterone and * - samples incubated with [ 4-ltfC] pregnenolone;. FIGURE 16. The morphology of adrenal c o r t i c a l c e l l s i n FCS-medium (a-c) and i n HS-medium (d-f) after up to 3 passages, phase optics, X 210. a. adrenal c o r t i c a l c e l l s i n FCS-medium sub-cultured from adrenal e p i t h e l i a l - l i k e monolayers i n f i r s t passage. C e l l s have developed a bipolar morphology. b. adrenal c o r t i c a l c e l l s i n FCS-medium sub-cultured from monolayers of adrenal e p i t h e l i a l - l i k e c e l l s i n second passage. Cell s s t i l l r e t a i n a b i l i t y to develop a bipolar morphology i n FCS-medium. c. adrenal c e l l s i n FCS-medium subcultured.from adrenal e p i t h e l i a l - l i k e monolayers i n their t h i r d >passage Cells appear more spread and not as bipolar as i n (a) and (b). d. adrenal c o r t i c a l c e l l s i n HS-medium sub-cultured from adrenal f i b r o b l a s t - l i k e monolayers i n firsts-passage. C e l l groups are cohesive and c e l l s appear epith-e l i a l - l i k e . e. adrenal c o r t i c a l c e l l s i n HS-medium subcult-ured from cultures of adrenal f i b r o b l a s t - l i k e c e l l s i n their second.passage. Cells are s t i l l able to develop an epith-e l i a l - l i k e configuration. f. adrenal c o r t i c a l c e l l s i n HS-medium subcultured from cultures of adrenal f i b r o b l a s t - l i k e monolayers i n their t h i r d passage. Cells appear flattened and not as cohesive as i n (d) and (e). 9 4 95 ( T a b l e 15 )\ b. £dd_i_tion_of_ HS-medium ^o_ad_re_na_l_f i^br_oW^s^-lLike_ce_lJLs_ The addition of HS-medium to secondary monolayers of adrenal f i b r o b l a s t - l i k e c e l l s had no influence on culture morphology or fluorogenic s t e r o i d production. A f t e r 3 days, however, the amount of histochemically demonstrable MECM appeared to be l e s s than i n c e l l s maintained i n FCS-medium. c. a^dd_i_tion_of_ F_CjJ-medium_to_ adr_enal_ eyjithel_ia_l-^like ^el ^ l s The addition of FCS-medium caused morphologic modulation as demonstrated by time lapse cinemicrography, from an e p i t h e l i a l - l i k e configuration to a f i b r o b l a s t - l i k e morphology, the main features of which are documented i n Figure 17. There was very l i t t l e c e l l move-ment i n cultures maintained i n HS-medium (Table 19K). Several to 8 h after the addition of FCS-medium, increased undulation of c e l l membranes was observed and then c e l l s began to move a c t i v e l y and by 8-15 h had become spindle shaped (Fig. 17b) and had begun to migrate away from one another. Fluorogenic st e r o i d production remained high(Table 16) for the f i r s t 8 h of exposure to FCS-medium and a trace of metachromasia was d i s c e r n i b l e i n cultures fixed at th i s time. By 20 h (Fig. 17c), steroid production had dropped by a factor of 7, m i t o t i c f i g u r e s i n c r -eased and production of MEjSM-v'.hadJ--:inG-i?eais'ed-'} (Table 16). At t h i s time, c e l l s were a c t i v e l y migrating upon the substratum (Table .19) • A f t e r 28-40 h i n the presence of FCS-medium (Fig. 17dye), s t e r o i d production had dropped by several orders of magnitude, the m i t o t i c index remained high and large amounts of MECM surrounded and covered a l l c e l l groups (Table 16, Fig.19a). Cultures at t h i s time resembled f i b r o b l a s t - l i k e monolayers o r i g i n a l l y grown i n FCS-medium (Fig. 5b). S i m i l a r l y , [4- 1 4C]pregnenolone metabolism al t e r e d when adrenal Table 16. The Effect of Fetal Calf Serum on the Phenotypic Expression of Adrenal E p i t h e l i a l Cells in- Vitro. Hours After Addition of Medium Supplement Fetal Calf Serum C e l l Morphology Fluorogenic Steroid Production 1 ug/24h/106 Cells Presence of Metachromasia Mitotic Index 2 0 e p i t h e l i a l 5.8 ± 1.22 None <1 8 transition;to f i b r o b l a s t - l i k e 3.4 ± 0.56 + <1 20 f i b r o b l a s t - l i k e 0.86± 0.37 + 5.0 ± 1.24 28 f i b r o b l a s t - l i k e 0.09+ 0.06 ++ 49.66 ± 3.60 40 f i b r o b l a s t - l i k e 0.01± 0.005 +++ 54.0 ± 9.2 48 f i b r o b l a s t - l i k e 0.01± 0.005 +++ 52.1 ± 2.3 Each value represents the mean of 6 cultures. The c e l l number increased during the course of the experiment from approximately 2x10^ to 2xl0 5 c e l l s per p e t r i dish. Steroid production was therefore standardized per 106 c e l l for comparative purposes. Means ± S.E.M. . Based on t o t a l of 8,000 c e l l s (6 cultures) and expressed as the number of mitotic figures per 1000 c e l l s ± S.E.M. FIGURE 17. Effects of FCS-medium on culture morphology of adrenal e p i t h e l i a l - l i k e c e l l s , phase optics, X 210. a. secondary adrenal culture grown with HS-medium showing e p i t h e l i a l type c e l l growth, prominent r e t r a c t i l e l i p i d inclusions. b. same colony as i n (a) but 8 h after the addition of FCS-medium. Cells have begun to separate from one another and are showing active membrane move-ment at this time. c. same area:but 24 h after substitution of FCS-medium for HS-medium, l i p i d inclusions are less num-erous and c e l l s are f i b r o b l a s t - l i k e and have begun to mig-rate away from one another. d-e. same area as (c) but 36 h(d) and 48 h(e) after substitution of FCS-medium for HS-medium: rapid c e l l growth, complete disappearance of l i p i d and a d i s t i n c t i v e bipolar morphology are observed. 99 e p i t h e l i a l - l i k e c e l l s were exposed to FCS-medium. Within 24 h aft e r exposure to FCS-medium, monolayers no longer produced detectable amounts of [4- 1^C]20a-hydroxypregn-4-en-3-one or [4- 1 1 +C]deoxycortico-sterone. In add i t i o n , pregn-5-ene-33,20ct-diol formation was reduced. By 72 h, the pattern of [4- 1 1 +C]pregnenolone metabolism by modulated adrenal f i b r o b l a s t - l i k e c e l l s was in d i s t i n g u i s h a b l e from adrenal f i b r o b l a s t - l i k e c e l l s o r i g i n a l l y grown i n FCS-medium (Table 17.) . B. Addition of 6-diazo-5-oxo-L-norleucine (DON) with FCS-medium  to adrenal e p i t h e l i a l - l i k e c e l l s The p o s s i b i l i t y that the regression of adrenal c o r t i c a l pheno-type observed i n FCS-medium was mediated by MECM production was considered because MECM production coEreU^ateiiiwel"-!--wi-th- the modulation of e p i t h e l i a l - l i k e c e l l s to a f i b r o b l a s t i c form and function i n FCS-medium and since MECM has been implicated i n a v a r i e t y of morphogen-e t i c phenomena (44-46') ^ .f) ^Ther:e#ore^>jfehe--response of adrenal e p i t h -e l i a l - l i k e c e l l s to FCS-medium i n the absence of MECM production was examined by exposing monolayers to FCS-medium and 6-diazo-5-oxo-L-norleucine (DON). The biochemical action of DON. has been well doc-umented.^ 1014) . DON blocks u t i l i z a t i o n of L-glutamine i n the trans-amidination reactions and thus i n t e r f e r e s with the biosynthesis of ..glycosaminoglycans and purines (104) . DON combines i r r e v e r s i b l y with the enzyme necessary f o r the formation of 5-phosphoribosylamine and blocks i t s conversion to formylgycinaminide ribonucleotide,sa>vital step i n purine synthesis probably r e s u l t i n g i n the i n h i b i t i o n of E. C o l i and mammalian c e l l growth'observed i n the presence of t h i s drug. DON has also been reported to i n h i b i t the s p e c i f i c transaminidase catalyzing the transfer of amide nitrogen from L-glutamine to fructose-Table 17. A l t e r a t i o n of [4- 1 4C] .Pregnenolone Metabolism After the Addition of FCS-Medium to Adrenal E p i t h e l i a l - l i k e C e l l s . Time After Addition of FCS Supplements (days) % T.4-14C] Pregnenolone Metabolism 1 Pregnenolone pregn-5-ene-3g, 20a-diol 20a-hydroxy-pregn-4-en-3 -one deoxycort-icosterone corticosterone 0 87 <v ± 2 . 8 9 6.6 ± 1 . 2 0.78 ± 0.47 0.87±.0 0.00 24 97.84 ± 0.09 0.76 ± 0.34 0.0 0.00 0.00 72 99.70 ± 0.10 0.21 ± 0.05 0.0 0.00 0.00 Cultures were incubated with 200,000/dpm [4- l 4C] pregnenolone for 8 h.. Values represent the mean of 4 cultures ± S.E.M. Table 18. The Effect of DON on the Fluorogenic Steroid Production, C e l l Morphology and MECM Production of Adrenal E p i t h e l i a l - l i k e Cells Exposed to FCS-Medium. Experiment # •Experimental Treatment 1 C e l l Morphology Fluorogenic Steroid Production ug/24h /10 6 c e l l s 2 Presence of Metachromasia 1 control HS-medium e p i t h e l i a l - l i k e 1.30 ± 0.49 None •HS-medium plus DON (50ug/ml medium) e p i t h e l i a l - l i k e 2.76 ± 1.32 None 10%FCS-medium plus D0N(50ug/ ml medium) e p i t h e l i a l - l i k e 0.20 ± 0.07 None 10%FCS-medium f i b r o b l a s t - l i k e trace ++ 2 10%FCS-medium f i b r o b l a s t - l i k e trace ++ 10%FCS-medium, D0N(50ug/ml) e p i t h e l i a l - l i k e 0.2 ± 0.009 None 10%FCS-medium, DON(50ug/ml), glucosamine (.1 mg/ml) fi b r o b l a s t - l i k e 0.1 ± 0.04 + Cultures were exposed to DON i n FCS-medium for a t o t a l of 24 h prior to c o l l e c t i o n of endogenous steroid i n HS-medium. Each value represents the mean of 6 cultures ± S.E.M. 102 6-phosphate for the synthesis of glucosamine, a precursor of glycos-aminoglycan synthesis (.105) • In contrast to cultures i n FCS-medium only, secondary cultures of adrenal e p i t h e l i a l - l i k e c e l l s exposed to 30-100yg DON/ml of FCS-medium for 24 h, did not develop a bipolar morphology and did not migrate away from one another but remained as c e l l groups (Fig. 18). Cel l s appeared to separate from one another somewhat and, as i n cultures i n FCS-medium without DON, an increase i n membrane r u f f l i n g and a samll increase i n c e l l movement within c e l l groups (Table l.o) was observed although migration did not occur. Since DON does not completely i n h i b i t glycosaminoglycan synthesis (.106), such movement i s l i k e l y the resu l t of small histochemically undetectable amounts of MECM. Cells that were isolated from one another due to group re t r a c t i o n , remained stationary, exhibiting r u f f l i n g movements around the entire c e l l periphery. MECM was not demonstrated histochemically at concentrations of 30-100yg DON/ml FCS-medium (Fig. 19d,e> and Table l'S). Steroid production dropped to about 1/6 of control e p i t h e l i a l - l i k e c e l l s i n HS-medium but remained higher than that of c e l l s exposed to FCS-medium only. DON added to control cultures i n the absence of FCS-medium did not have a si g n i f i c a n t effect on fluorogenic steroid production (Table 18). Adrenal e p i t h e l i a l - l i k e c e l l s exposed to lyg DON/ml of FCS-medium developed a f i b r o b l a s t - l i k e morphology and produced MECM (Fig. 19a). Cel l s exposed to 15yg DON/ml FCS-medium were also f i b r o b l a s t - l i k e although c e l l s appeared more cohesive and did not produce detectable MECM (Fig. 19b). Tha • ;.(":.icr. : i c The addition of O.lmg glucosamine/ml FCS-medium, lmg glutamine FIGURE 18. The' effect of S.0pg/iml'-6f-o6.Tdiazor-5-r.oxo^L-norleucine .(iDON)l added 'wi-th^EGSsmediumconuthe .• eultureomorphology±of adrenal e p i t h e l i a l - l i k e c e l l s , phase optics, X 200. a. secondary culture of adrenal e p i t h e l i a l - l i k e c e l l s grown i n HS-medium. b-re. same area as i n (a) but 4 h (b), 10 h (c) , 24 h (d) and 48 h (e) l a t e r . Although groups, appear less organized than i n (a), c e l l s generally have not developed a f i b r o b l a s t - l i k e morphology, have remained cohesive and have not migrated away from one another. Table 19. The E f f e c t of Serum, DON and Mo d i f i e d Substrata on Adrenal C o r t i c a l C e l l Movement. Substratum Morphology P r i o r O r i g i n a l Serum Treatment Morphology A f t e r Rate of Movement Metachromasia to Treatment Supplement (24h) Treatment Units/24 h 1 A. Tissue ( Culture e p i t h e l i a l - l i k e HS-medium e p i t h e l i a l 0.22 ± 0.05 e p i t h e l i a l - l i k e HS-medium 10% FCS-medium f i b r o b l a s t - l i k e 5.75 ± 0.47 ++ e p i t h e l i a l - l i k e • HS-medium 10% FCS-medium e p i t h e l i a l - l i k e 0.92 ± 0.17 -p l u s ^ O ug/ml DON e p i t h e l i a l - l i k e HS-medium 10% FCS-medium f i b r o b l a s t - l i k e 4.8 ± 0.45 + plus 50 ug/ml DON plus 0.1 mg/ml gluco-amine f i b r o b l a s t - l i k e 10%FCS-medium 50ug/ml DON f i b r o b l a s t - l i k e 4.0 ± 0.35 + B. Treated Growth Surfaces sulphon- f i b r o b l a s t - l i k e . HS-medium f i b r o b l a s t - l i k e 2.4 ±. 0.32" + ated p o l y - l y s i n e f i b r o b l a s t - l i k e HS-medium — f i b r o b l a s t - l i k e 2.1 ± 0.17 -t r e a t e d brushed e p i t h e l i a l - l i k e . HS-medium - e p i t h e l i a l - l i k e 1.0 ± 0.25 -t i s s u e c u l t u r e U n i t s are given i n cm, measured on a moviscope screen and have been magnified by 70 times the a c t u a l d i s t a n c e t r a v e l l e d by c e l l s / 2 4 h . FIGURE 19. Adrenal e p i t h e l i a l - l i k e c e l l s exposed to DON and FCS-medium. Cultures were stained with toluidine blue at pH 2.5, X 210. a. cultures exposed to FCS-medium only, ;jshowing large amounts of MECM. b. cultures exposed to 15ug DON/ml FCS-medium. Cells are f i b r o b l a s t - l i k e but no detectable MECM/was observed. c. cultures exposed to 50ug DON and. O.lmg gluc-osamine/ml FCS-medium. Cells are f i b r o b l a s t - l i k e and MECM i s evident. d. cultures exposed to 30yg DON/ml FCS-medium. Cells appear e p i t h e l i a l - l i k e and do not produce detectable MECM. e. cultures exposed to 50ugDON/ml FCS-medium. Cells appear e p i t h e l i a l - l i k e and more cohesive than i n (d). No MECM i s observed. 107 /ml FCS-medium or O.lmg glucosamine-6-phosphate/ml FCS-medium to adrenal c e l l s exposed to 50|Jg DON/ml FCS-medium p a r t i a l l y reversed the effects of DON i n that c e l l s developed a f i b r o b l a s t - l i k e morphology (Fig. 19c), produced trace amounts of metachromatic ext r a c e l l u l a r matrix (Fig. 19c and Table 18) and showed a si g n i f i c a n t increase i n the rate of c e l l movement (Table 19)(P<0.001). Furthermore, c e l l s were able to p u l l away from one another and, when th i s occurred, were able to trans-locate on the substratum although frequently changing direction. Steroid production was not s i g n i f i c a n t l y d ifferent from that of c e l l s exposed to DON i n FCS-medium but remained higher than that of c e l l s exposed to FCS-medium only (Table 18). C. Addition of glycosaminoglycans to adrenal c o r t i c a l e p i t h e l i a l - l i k e c e l l s i n HS-medium The a b i l i t y of DON to influence regression of adrenal c o r t i c a l e p i t h e l i a l - l i k e c e l l s exposed to FCS-medium strongly suggested that onset of MECM production was responsible for the observed a l t e r a t i o n of growth'patterns. Furthermore, histochemical data suggested that hyaluronic acid might be a component of the MECM produced by adrenal c o r t i c a l c e l l s i n the presence of FCS-medium. Therefore, the effect of adding^ exogenously, the glycosaminoglycans,hyaluronic acid and chondroitin sulphate were examined. Adrenal c o r t i c a l e p i t h e l i a l - l i k e c e l l s maintained i n HS-medium developed a more angular morphology, intermediate between a r a d i a l l y symmetrical and a bipolar configuration when exposed to 50-500ug/ml HS-medium of hyaluronic acid. MECM was not produced i n the presence of hyaluronic acid i n HS-medium. Cells remained cohesive and did not appear to migrate away from one another. There was no increase i n mitotic a c t i v i t y . C e l l s produced small amounts of fluorogenic 109 ste r o i d i n comparison to c o n t r o l cultures of adrenal e p i t h e l i a l - l i k e c e l l s i n HS-medium (Fig. 5c and Table20 •) • The addition of l-100yg chondroitin sulphate/ml HS-medium had no e f f e c t on culture morphology or ster o i d production (Table 20). D. Growth surfaces In order to determine whether the charge density and/or fibrous nature of glycosaminoglycans such as hyaluronic acid and endogenous MECM were responsible f o r the e f f e c t of these molecules on adrenal c o r t i c a l c u l t u r e morphology and c y t o d i f f e r e n t i a t i o n , c e l l s were grown on: 1. sulphonated (negatively charged) surfaces; 2. p o l y l y s i n e treated ( p o s i t i v e l y charged) surfaces; 3. mechanically etched surfaces and 4. n i t r i c acid etched surfaces. The e f f e c t of growth on these surfaces i n HS-medium on s t e r o i d production, growth patterns and motile behavior were then examined, a. ^ulp_honajted_ _sur_f a_ce_s_ i ) estimation of the degree of sulphonation Sulphonation of polystyrene surfaces at 55°C, as estimated by dye binding, increased l i t t l e up to 3 minutes of treatment and then exponentially up to ^h, leveled o f f between { to 5h and appeared to increase again by 10 h treatment (lElg,?t-J2QA*') • Decreasing the pH of the dye so l u t i o n from 5.0 to 1.0, decreased dye binding s l i g h t l y (Table 2/]) suggesting that at a higher pH, some non-specific binding loccurred and i n d i c a t i n g that dye binding should be estimated at lower pH. i i ) c e l l attachment to sulphonated surfaces C e l l "adhesion", measured as the percent of attached c e l l groups able to withstand inversion of aiicul.t-ureizd-i'sh 5 min a f t e r seeding, increased with increasing sulphonation of surfaces i n both HS-medium, Table.--20. The Influence of Added Glycosaminoglycans on Steroid Production, Culture Morphology and MECM Production of Adrenal E p i t h e l i a l - l i k e C e l l s . Glycosaminoglycan 1 Culture Morphology Steroid Production2 ug/105 cells/24 h Metachromasia Mitotic Index 2 (per 1000 c e l l s ) control e p i t h e l i a l - l i k e 3.69 ± 0.81 None <1 hyaluronic acid intermediate trace None <1 chondroitin sulphate e p i t h e l i a l - l i k e 4.68 ± 1.79 None <1 Hyaluronic acid and chondroitin sulphate (50 ug/ml medium) were added to cultures of differentiated e p i t h e l i a l - l i k e c e l l s for 24 h prior to steroid determination. Values based on a t o t a l of 8 cultures. Means ± S.E.M. Table 21. Influence of pH of Toluidine Blue Solution . on the S p e c i f i c i t y of Staining on Sulphonated Dishes. pH of Dye Solution Molarity of Dye Solution mg of Bound to Treated 15 min. Toluidine Blue Surfaces After: 30 min. 1.0 0.0001 0.044 0.044 2.5 0.0001 0.061 0.075 5.0 0.0001 0.073 0.089 ' 112 F I G U R E 2 0 A . S u l p h o n a t i o n of g r o w t h s u r f a c e s at 55°C a n d t h e effect c e l l a t t a c h m e n t — i 1 1 1 — 1— 1 1 0.02 0.05 0.1 0.2 1.0 2.0 5.0 HOURS OF TREATMENT WITH SULPHURIC ACID EPITHELIAL-LIKE INTERMEDIATE FIBROBLAST-LIKE CELL MORPHOLOGY IN HS " MEDIUM F I G U R E 2 0 B . E f f e c t o f g l u t a r a l d e h y d e f i x a t i o n o n c e l l a t t a c h m e n t o n c h a r g e d s u r f a c e s . 100-, 804 5 6 0-x o £ 40-LU o 204 UNFIXED C E L L S IN HANKS B S S PETRI DISHES SULPHONATED SURFACES POLYLYSINE TREATED SURFACES GLUTARALDEHYDE FIXED C E L L S IN HANKS B S S 114 and Waymouth medium only (Fig.20A) to a maximum of 90% attachment on surfaces t r e a t e d f o r 30 min w i t h s u l p h u r i c a c i d . C e l l attachment decreased to 30% on surfaces t r e a t e d f or lOh. C e l l attachment was g e n e r a l l y lower i n HS-medium although the trend of i n c r e a s i n g a t t a c h -ment w i t h i n c r e a s i n g sulphonation was s i m i l a r to that observed i n serum f r e e medium. C e l l morphology i n HS-medium g e n e r a l l y remained e p i t h e l i a l - l i k e on surfaces t r e a t e d f o r up to 5 min w h i l e c e l l s p l a t e d onto surfaces t r e a t e d f o r .5915 min were of intermediate morphology, and c e l l s p l a t e d onto surfaces t r e a t e d f o r j-5 h were f i b r o b l a s t - l i k e . C e l l s d i d not s e t t l e on surfaces treated f o r 10 h.'V^ Eigv 20A). C e l l s kept at 4°C f o r 1 h p r i o r to seeding showed a s i m i l a r (Fig.20A) increase i n c e l l attachment w i t h increased sulphonation of growth surfaces but percent attachment was s e v e r a l times lower than that of warm c e l l s . ( C e l l s f i x e d w i t h glutaraldehyde d i d attach-ttohpolysine 'ereatedhandtpetriLrddl^hes (but7.notv'to. sulphonated surfaces ( F i g . 20B). i i i ) c u l t u r e morphology, c e l l movement and f l u o r o g e n i c s t e r o i d  production 1. HSj^med ium Adrenal c o r t i c a l c e l l s subcultured mechanically as c e l l groups from confluent primary c u l t u r e s and grown on sulphonated surfaces were u n l i k e c e l l s grown on t i s s u e c u l t u r e surfaces i n that they were f i b r o b l a s t - l i k e ( F i g . 5d), e x h i b i t e d s i g n i f i c a n t l y (P<0.001) more movement and produced only t r a c e amounts of f l u o r o g e n i c steroid„i(Table lf9,2?!>. The v i a b i l i t y of adrenal c o r t i c a l c e l l s grown on sulphonated surfaces was confirmed by responsiveness to ACTH (Table 2.2) and by v i s i b l e l a c k of c e l l n e c r o s i s a f t e r maint-eriancee of sample c u l t u r e s f o r 3 weeks. Fragments obtained by ' r-r>c\..\ 1 'rcr.e, 1 rc^-jior?holc3,i ' a l l y vni toim* ~*c i.e.." v . . Table 22. The Influence of Treated Growth Surfaces on the Steroid Production, MECM Production and Culture Morphology of Adrenal Co r t i c a l E p i t h e l i a l - l i k e Cells i n HS-medium. Treatment of Growth Surface mg of Bound Toluidine Blue Culture Morphology Fluorogenic Steroid Production of ug/24h/106 c e l l s Metachromasia Mitotic, per 1000 Index c e l l s Basal ACTH,100mU None 0.005 ± 0.001 e p i t h e l i a l - l i k e 2.810.81 25.64±2.89 None <1 Sulphuric acid (30 min.@55°C) 0.11 :± 0.0054 f i b r o b l a s t - l i k e 0.005 0.05 ±0.01 None <1 Poly-lysine treated 0.00 f i b r o b l a s t - l i k e 0.005 Not assayed None <1 Mechanically brushed 0.005 ± 0.001 e p i t h e l i a l - l i k e 0.4 Not assayed None <1 N i t r i c Acid treated (30min.@55°C) 0.005 ± 0.00 e p i t h e l i a l - l i k e Not assayed Not assayed None <1 1 Values represent the means ± S.E.M..of 8 cultures. mechanical subculture from morphologically uniform secondary cultures ± J- D of e p i t h e l i a l - l i k e c e l l s were also plated onto both untreated and sulphonated surfaces i n HS-medium. A l l c e l l groups plated onto tissue culture dishes remained e p i t h e l i a l - l i k e while c e l l groups plated onto sulphonated surfaces changed to a f i b r o b l a s t - l i k e morph-ology, suggesting that growth on sulphonated surfaces was modifying the e p i t h e l i a l configuration rather than selecting for another c e l l type. 2. FjC^-medium Cells grown on sulphonated surfaces i n FCS-medium were f i b r o b l a s t -l i k e and exhibited s i g n i f i c a n t l y fewer mitotic figures than those grown on tissue culture surfaces i n FCS-medium. Even i n the presence of FCS-medium, MECM was not produced when c e l l s were grown on sulphonated surfaces (Table 2.§) . b. p_olyl^^ine_t r_ea^ t e_d_su_r f_ac_es^  i ) c e l l attachment Fixed and l i v e adrenal c o r t i c a l c e l l s attached equally well to polylysine treated surfaces,(Fig. 20B). i i ) culture morphology, c e l l movement and fluorogenic steroid  production Adrenal c o r t i c a l c e l l s subcultured mechanically as c e l l groups from confluent primary cultures and grown on polylysine treated sur-faces i n HS-medium were f i b r o b l a s t - l i k e (Fig. 5e) and produced only trace amounts of fluorogenic steroid (Table 22). No MECM was dem-onstrated. Cel l s were able to move on thi s substratum i n that the rate of.movementewa'SLtsignificant;ly,agreate'f<(iE<$U001}iathan thatzgof,xeelis grown on tissue culture growth surfaces (Table 1-9). Although c e l l s appeared less cohesive than those grown on sulphonated surfaces (Fig. 5d;,e) ) Table 23. Mi t o t i c Index and Metachromatic Ext r a c e l l u l a r Matrix Production of Adrenal C o r t i c a l Cells Grown On Sulphonated Surfaces i n FCS-medium. Growth Surface C e l l Morphology M i t o t i c Index''' M i t o t i c Figures/ 1000 Cells Metachromasia Tissue Culture Sulphonated f i b r o b l a s t - l i k e f i b r o b l a s t - l i k e 66 ± 2.0 8.2 ± 1.3 ++ None 1 Values represent means determined from 4 cultures. Means±S.E.M. they were not able to translocate i n one direction as could c e l l s grown on either sulphonated surf aces i n HS-medium or on tissue cultures surfaces i n FCS-medium. Rather, they appeared to move back and for t h as i f strongly attached to the substratum, c. et_che_d_surf_ac_es_ In order to determine whether the development of a f i b r o b l a s t -l i k e morphology on charged surfaces resulted from contact guidance (356) due to physical a l t e r a t i o n of growth surfaces upon stringent treatment ( i e . exposure to sulphuric acid for 30 min at 55 C) rather than to increased surface charge density per sej'j adrenal e p i t h e l i a l -l i k e c e l l s were grown on mechanically brushed and n i t r i c acid etched surfaces and morphology and steroid production determined, i ) brushed surfaces 1. c e l l _ a t tachment Adrenal c o r t i c a l c e l l s did not attach more rapidly to brushed surfaces than to untreated tissue culture surfaces. 2. culture morphology^ cel_l_mc^vemen_t and steroid production „-I- alCells' grown on brushed surfaces remained as cohesive groups a l -though colonies tended to have a ragged appearance and-to l i n e up along the more obvious grooves (Fig. 5f). Cells at the edges of the colony tended to a l i g n themselves along grooves and i n some cases pulled away from the main c e l l group and moved along the grooved areas. C e l l movement within a c e l l group was s i g n i f i c a n t l y greater on brushed surfaces i n HS-medium than on untreated tissue culture surfaces i n HS-medium (Table 119) (P<0.001) and r u f f l i n g of c e l l mem-branes was marked compared to c e l l s grown on untreated surfaces. MECM was not detected. Fluorogenic steroid production dropped by a factor of 5 compared to e p i t h e l i a l - l i k e c e l l s grown on untreated tissue culture surfaces ; (Table 22). i i ) n i t r i c acid treated surfaces 1. ceT l_a_tta^chment Adrenal c o r t i c a l c e l l s did not attach more rapidly to n i t r i c acid treated surfaces than to untreated tissue culture surfaces. 2. ^el_l_mc^rpJio_lc^gy_ Adrenal c o r t i c a l c e l l s grown on n i t r i c acid etched surfaces remained e p i t h e l i a l - l i k e and were indistinguishable from control cultures of e p i t h e l i a l - l i k e c e l l s grown on untreated tissue culture surfaces (Table 22.). d. ^^PB-lBB—^l^SPlPB. ™^ £.r£.s£.0P_y_0£. grwj^h_surf_aces_ i ) sulphonated surfaces Examination of sulphonated surfaces and tissue culture growth surfaces with scanning electron microscopy revealed no difference i n topography (Fig. 21a,b) Both surfaces were thrown into shallow ridges of less than O.lu i n width. These ridges occurred as a mesh-l i k e network. i i ) brushed surfaces The grooves distinguishable on brushed surfaces using l i g h t microscopy were shown on scanning electron microscope^to b_e?roughened and jagged (Fig. 21c). i i i ) n i t r i c acid treated surfaces N i t r i c acid treated surfaces were v i s i b l y altered and could be seen to be etched using l i g h t microscopy. Examination of the surface with scanning electron microscopy revealed an increase i n the number and width of grooves (Fig. 21d). E. Mechanical dissociation of adrenal c o r t i c a l c e l l s In order to examine the effect of altered growth patterns on FIGURE 21. Scanning electron microscopy of growth surfaces, X 10,000. a. untreated tissue culture growth surfaces contain small ridges. b. sulphonated surface where ridges appear i d e n t i c a l to (a). c. brushed surfaces appear roughened and jagged. d. n i t r i c acid treated surface shows a s l i g h t l y greater number of grooves and ridges than untreated growth surfaces i n (a). 121 endogenous steroid production i n the absence of MECM production or exogenous drugs, the fluorogenic steroid production of monolayer fragments disrupted by pipetting and seeded into HS-medium was examined. Settled c e l l s were f i b r o b l a s t - l i k e and did not produce MECM. Steroid production was lower than that of e p i t h e l i a l - l i k e c e l l s grown i n HS-medium but higher than that of c e l l s grown i n FCS-medium or on sulphonated surfaces (Table 2$). F. Colcemid Colcemid added at concentrations of 10 6M caused the develop-ment of an e p i t h e l i a l - l i k e morphology even when c e l l s were grown i n FCS-medium. Colcemid stimulated s l i g h t l y the metabolism of [4- l l +C]-pregnenolone to deoxycorticosterone and 20a-hydroxypregn-4-en-3-one but had not effect on the production of endogenous fluorogenic steroid production (Table 23'*,265) . Lumicolchicine had no effect on c e l l morphology or steroid production. 123 Table 24. The Effect of Mechanical Dissociation of Monolayer Fragments Obtained from Confluent Primary Cultures on C e l l Morphology, Fluorogenic Steroid Production and Production of MECM. Treatment of Monolayer Fragments C e l l Morphology Fluorogenic Steroid Production" yg/24h/106 Cells Meta-chromasia fragments i n t a c t ; seeded i n HS-medium e p i t h e l i a l - l i k e 1.35 ± 0.35 None fragments i n t a c t ; seeded i n FCS-medium f i b r o b l a s t - l i k e trace ++ fragments mechanic-a l l y dissociated; seeded i n HS-medium f i b r o b l a s t - l i k e 0.19 ± 0.07 None 1 Values based on 8 cultures. Mean ± S.E.M. CSl Table 25. E f f e c t of Colcemid on the [4- 1 4C] Pregnenolone Metabolism 1 of Adrenal C o r t i c a l F i b r o b l a s t - l i k e C e l l s . C u l t u r e Conditions Time of Exposure to Colcemid Colcemid Concentration (molar) % r a d i o a c t i v i t y 1 C o r t i c o s -terone pregnenolone pregn-5-en-38,20oi-diol 20ahydroxy-pregn-4-en-3-one deoxy-cortico-sterone 25% FCS 0 0 98.22 ± 1.20 1.39 ± 0.54 0.00 0.00 0100 2 days I O - 6 98.74 ± 0.26 0.53 ± 0.40 0.55 ± 0.55 0.44 ± 0.04 0.00 10% FCS 0 0 97.34 ± 1.62 0.50 ± 0.34 0.00 0.00 0.00 3 days io- 6 96.40 ± 1.52 1.98 ± 0.45 0.31 ± 0.31 0.36 ± 0.36 0.00 8 h 1 0 - 6 89.78 ± 3.12 8.18 ± 2.77 0.73 ± 0.43 0.38 ± 0.38 0.00 10% HS 0 0 97.81 ± 0.53 1.58 ± 0.50 0.07 ± 0.01 0.03 ± 0.002 0.00 8 h . 1 0 - 6 97.50 ± 0.54 1.76 ± 0.39 0.13 ± 0.12 0.02 ± 0.001 0.00 Values are based on 4 c u l t u r e s . Means ± S.E.M. Table 26. The Influence of Colcemid on Endogenous Steroid Production of Adrenal C o r t i c a l C e l l s . Exp. # Growth Surface C e l l Type and Culture Conditions Colcemid Concentration (Molar) Meta-chromasia Culture Morphology Steroid Production ug/24h/106 c e l l s l 1. tissue culture e p i t h e l i a l - l i k e , HS-medium 0 - e p i t h e l i a l - l i k e 6.43 ± 3.82 tissue culture e p i t h e l i a l - l i k e , HS-medium IO"6 - e p i t h e l i a l - l i k e 4.24 ± 1.92 2. tissue culture f i b r o b l a s t - l i k e FCS-medium 0 + f i b r o b l a s t - l i k e 0.15 ± 0.02 tissue culture f i b r o b l a s t - l i k e FCS-medium 10 - 6 - e p i t h e l i a l - l i k e 0.15 ± 0.05 3. tissue culture f i b r o b l a s t - l i k e FCS-medium 0 +++ f i b r o b l a s t - l i k e trace tissue culture f i b r o b l a s t - l i k e FCS-medium IO"6 ++ e p i t h e l i a l - l i k e trace 4. sulphonated f i b r o b l a s t - l i k e HS-medium 0 - f i b r o b l a s t - l i k e trace sulphonated f i b r o b l a s t - l i k e io- 6 - e p i t h e l i a l - l i k e trace 5. poly-lysine treated f i b r o b l a s t - l i k e f i b r o b l a s t - l i k e 0 10"6M _ f i b r o b l a s t - l i k e f i b r o b l a s t - l i k e trace trace 1 Values are based on 8 cultures. Means ± S.E.M. 126 DISCUSSION I. Origin and nature of secondary cultures of adrenal c o r t i c a l c e l l s A. The_ r_el_at_ionship_of_ sec.on.da.ry_ _adr_ena_L m^ onol^ ay_e_rs_t(__ p_revi_o_usly de__cr_ibecl c e l l types injr_imar^_c_jl_tur_e___ U n t i l recently,(54), the only available culture systems of adrenal cortex were a tumorigenic c e l l l i n e (67) and normal adrenal c e l l pop-ulations maintained i n primary culture (72-75). Although the tumor c e l l l i n e provides large homogeneous populations of functional c e l l s , i t exhibits abnormal steroid biosynthetic pathways and response to ACTH (67). Attempts to grow normal mammalian adrenal c o r t i c a l tissue in vitro have been made since the f i r s t development of tissue culture techniques (107). Adrenal c e l l s which exhibit a l l of the u l t r a s t r u c t -u r a l response to ACTH that are char a c t e r i s t i c of zona fasc i c u l a t a paren-chyma in vivo and which t y p i c a l l y display a highly d i s t i n c t i v e poly-gonal morphology (Fig. 2c), can be maintained i n primary tissue culture. However, thei r occurance i n very small numbers, as w e l l as the presence of other c e l l types i n these primary monolayers, makes biochemical ana-l y s i s d i f f i c u l t to interpret. Nevertheless, i n the past, the fluorogenic steroid produced by primary monolayers has been ascribed to these c e l l s assuming that the other c e l l types,pwhiehfinclude f i b r o b l a s t - l i k e as w e l l as lipid-containing c e l l s , represent non-endocrine, connective tissue elements only. Such an assumption i s apparently based on morphologic c r i t e r i a . This concept that c e l l morphology i s an inherent phenotypic ch a r a c t e r i s t i c of c e l l s and can be used as a r e l i a b l e c r i t e r i o n for i d e n t i -f i c a t i o n of tissue c e l l types such as functional parenchyma as compared 127 to "non-functional" connective tissue, i s largely derived from h i s t o l o g i c a l studies of c e l l s in vivo where environmental conditions are r e l a t i v e l y w e l l controlled. The advent of tissue culture techniques allowing mani-pulations of the microenvironment has offered a means with which to test this assumption. Generally, i t appears that c e l l s , at least of mesodermal o r i g i n , exhibit a large repertoire of morphologic forms ranging from epi-t h e l i a l - l i k e to f i b r o b l a s t - l i k e or bipolar forms. Although Garber, as early as 1953 (108) emphasized the transient nature of c e l l morphology i n tissue culture, i t i s only recently that alterations i n c e l l morphology are being considered as modulations i n shape and not necessarily the resul t of selection of different c e l l types. The demonstration of adrenal c o r t i c a l characteristics i n c e l l s that are morphologically i n d i s t i n g u i s h -able from connective and adipose tissue, which they have theretofore been regarded as, indicates the necessity to re-examine the c r e d i b i l i t y of cl a s s i f y i n g c e l l s according to their morphologic forms. Although secondary cultures of adrenal f i b r o b l a s t - l i k e and l i p i d -containing e p i t h e l i a l - l i k e c e l l s reported here and the polygonal adrenal c e l l s i n primary culture, a l l exhibit adrenal c o r t i c a l c h a r a c t e r i s t i c s , the differences i n their u l t r a s t r u c t u r a l response to ACTH are i n keeping with the p o s s i b i l i t y that they probably originate from different c e l l types in vivo. Apparently, the polygonal adrenal c e l l s i n primary culture represent fasciculata c e l l s that have been able to survive in vitro since t h e i r response to ACTH closely resembles that of f a s c i c -ular c e l l s in vivo. The c e l l u l a r o r i g i n within the adult adrenal cortex of the secondary adrenal monolayers described here i s not so obvious. Nevertheless, the following characteristics of these secondary monolayers, including corticosterone production, 128 steroidogenic and u l t r a s t r u c t u r a l response to ACTH as w e l l as culture homogeneity- combine to make this culture system more suitable for investigation of adrenal c o r t i c a l trophic response to ACTH than currently available c e l l lines (67) and primary culture techniques (72-75). B. Cellular o r i g i n of secondary a^ren^l_monqlayers_ wijth^n adult adren.al_ j;land. In the present study, c e l l s i n the e p i t h e l i a l - l i k e form resemble' parenchyma of the zona fas c i c u l a t a i n that they contain , large amounts of l i p i d depletable by ACTH (101), produced corticosterone i n the presence or absence of ACTH (109,110), and,,when maximally stimulated by trophic hormone, produced corticosterone i n amounts similar to the acute ACTH-stimulated f a s c i c u l a r zone i n vivo (23, 109). In addition, the increase, upon stimulation with ACTH, i n SER, polysome formation and surface micro-v i l l i i s t y p i c a l of some of the u l t r a s t r u c t u r a l changes observed i n f a s c i c -ular c e l l s i n vivo (101,111). However, s i g n i f i c a n t differences e x i s t between the e p i t h e l i a l - l i k e c e l l s and adult zona fasciculata c e l l s described in vivo (101,111). These include high corticosterone production i n the absence of ACTH, rapid trophic steroidogenic response to ACTH and lack of mitochondrial changes i n the presence of ACTH. The production of several yg of corticosterone/10 6 cells/24h i n the absence of ACTH i s much higher than that of fascicular c e l l s i n the hypophysectomized rats where steroid production drops to 1/10 of this amount (109,110). Rather, t h i s production of corticosterone i s comparable to that of fas c i c u l a r c e l l s i n the unstressed intact rat (23). Furthermore, the a b i l i t y to respond maximally to ACTH after only 3 days of treatment represents a trophic response that 129 i s considerably more rapid than that observed in vivo (112). Zona f a s c i c -ulata c e l l s i n the unstressed animal are unique i n that mitochondrial cristae are vesicular (101,111). Although t h i s ultrastructure disappears upon hypophysectomy or growth i n primary culture i n the absence of ACTH, the addition of ACTH in vitro restores the vesicular appearance of the mitochondrial cristae (72,75) within 3 days. The most marked difference between the adrenal c o r t i c a l c e l l s described here and fascicular c e l l s in vivo or i n primary culture i s the lack of such mitochondrial changes. This Indicates either that culture conditions are preventing such' a', transformation or that the c e l l s described here do not originate from the c e l l s of the fascicular zone but represent another c e l l type. In the investigations of Kahri (72) and others (73-75) c e l l s that are able to exhibit mitochondrial changes co-exist with those that do not (72), strongly suggesting that culture conditions are not the l i m i t i n g factor. The p o s s i b i l i t y that c e l l s might derive from glomerulosa c e l l s , which in vivo generally, do not respond morphologically to ACTH (101,111), i s unlikely since glomerulosa c e l l s have been shown to exhibit mitochon-d r i a l changes i n response to ACTH. in vitro and, upon excessive stim-u l a t i o n , in vivo (113). I t should be pointed out, as a matter of in t e r e s t , that the emphasis placed upon the importance of mitochondrial cristae changes to subsequent corticosterone production are probably u n j u s t i f i e d since i n the case reported here, adrenal e p i t h e l i a l - l i k e c e l l s were able to produce corticosterone i n amounts comparable to maximally stimulated fascicular c e l l s in vivo, but i n the absence of mitochondrial changes„(23, 109) (Table 11). Secondary cultures of adrenal e p i t h e l i a l - l i k e c e l l s resemble most closely, the f e t a l rat adrenal cortex prior to 16.5 days of development which at this time contains l i p i d inclusions, small amounts of RER and 130 SER and mitochondria with lamellar cristae (111). Furthermore, Roos (114) has shown that the f e t a l adrenal cortex of this stage i s capable of making coricosterone i n amounts comparable to c e l l s i n the unstressed intact adult rat despite the lack of large amounts of SER and absence of ACTH production in vivo at this time (23). Unfortunately t h i s resemb-lance does not indicate the c e l l u l a r o r i g i n i n the adult cortex, although i t raises the p o s s i b i l i t y that the c e l l s described here arise from a multi-potentiaiL or stem c e l l , a p o s s i b i l i t y that i s p a r t i c u l a r l y a t t ractive since the e p i t h e l i a l - l i k e c e l l s can be forced to regress to a f i b r o b l a s t -l i k e c e l l which i s morphologically si m i l a r to capsular fibroblasts i n that ifc.egon-t'aiiisnlarge amounts of microfilament bundles, dilat e d RER and prod-uce basement membrane-like material which i s closely applied to the entire c e l l surface separating the c e l l s from one another (115). The possible o r i g i n from the capsule of the adrenal c o r t i c a l c e l l s described here i s of p a r t i c u l a r interest i n view of the implicated, a l b e i t controv-e r s i a l , role, of capsular c e l l s as stem c e l l source during adrenal c o r t i c a l regeneration (116-120) and of the mesodermal derivation of the adrenal cortex (103). C e l l forms within the capsule that ware intermediate be-tween connective tissue and glomerulosa c e l l s i n terms of l i p i d content, c e l l morphology and SER have been described at the hi s t o l o g i c (117,118')-and electron microscopic l e v e l (119). Furthermore, the demonstration that mitochondrial and SER changes i n capsular c e l l s , i d e n t i f i e d by the presence of RER and location within the capsule, occurred, during adrenal c o r t i c a l regeneration lend c r e d i b i l i t y to former observations (119). In addition, upon administration of ACTH i n massive doses, for several weeks, the appearance of glomerulosa c e l l s within the capsule has been noted (120). However, most investigators refute the potential role of capsular c e l l s as stem c e l l s since i n the vast majority of studies, forms 131 intermediate between capsular and parenchymal c e l l s are not observed Y (102,121) and i t i s g e n e r a l l y thought that glomerulosa c e l l s provide a source of new parenchymal c e l l s during adrenal regeneration (113). The discrepancy ^ between;:these and e a r l i e r r e s u l t s has not been resolved" but rcould r e s u l t from the methods used to study regeneration. I f , a f t e r e n u c l e a t i o n of the inner cortex, a considerable number of parenchymal c e l l s were l e f t behind, c o r t i c o s t e r o n e synthesis might continue i n high enough l e v e l s so as to prevent a massive r e l e a s e of ACTH. I f , however, most parenchymal c e l l s were removed, c o r t i c o s t e r o n e production would drop to n e g l i g i b l e amounts and ACTH l e v e l s would increase and remain hig h u n t i l s u f f i c i e n t c o r t i c o s t e r o n e were produced to act upon the p i t -u i t a r y (23). In the presence of such hi g h l e v e l s of ACTH, capsular c e l l s might be forced to transform to parenchymal c e l l s (120). Such s p e c u l a t i o n i s p a r t i c u l a r l y e n t i c i n g i n view of the l o s s of connective t i s s u e p r o p e r t i e s and assumption of cftvert adrenal c o r t i c a l c h a r a c t e r i s t i c s upon exposure of the secondary c u l t u r e of adrenal f i b r o b l a s t - l i k e c e l l s reported here to what are p h y s i o l o g i c a l l y massive doses of ACTH in vivo (102). C. Is t_he pJi^no^tyj>^c_expr_es^si^on. ^^^an^^^^^^^^^^s^.^^^^ The a t y p i c a l morphology and low l e v e l of s t e r o i d production of secondary c u l t u r e s of adrenal c o r t i c a l c e l l s i n the f i b r o b l a s t - l i k e form suggest a l i m i t e d s t a t e of d i f f e r e n t i a t i o n or incomplete expression of the adrenal c o r t i c a l phenotype. The s t e r o i d production of these f i b r o b l a s t -l i k e c e l l s i s lower by 1-2 orders of magnitude than the s t e r o i d production of the adrenal e p i t h e l i a l - l i k e c e l l s whose s t a t e of c y t o d i f f e r e n t i a t i o n , i n terms of s t e r o i d p r o d u c t i o n , ifcgs more s i m i l a r to adult parenchyma-in vivo. 132 This difference i s reminiscent of stages i n embryonic development where the phenotypic expression of a variety of tissues i s preceeded by a stage at which the differentiated phenotype-is not maximally expressed though the c e l l s already synthesize t i s s u e - s p e c i f i c products at levels several orders of magnitude below those of the f u l l y developed tissue (14). This state, termed proto-differentiation by Rutter (14), was o r i g i n a l l y defined to describe a point during the temporal sequence of pancreatic develop-ment where mitosis was common and type specific-products were synthesized i n vefyeipwlamountsnusual'ly.lip^ i n the f u l l y cyto-d i f ferentiated state (14). Further study indicated an inertness to the physiological parameters con t r o l l i n g the degree of phenotypic expression i n the adult (17). For several obvious reasons i t i s not possible to d i r e c t l y compare the state of adrenal c o r t i c a l phenotypic expression with pancreatic development. Although d e t a i l s on early cytodifferentiation of the adrenal cortex are not available, other mesodermally derived tissues such as muscle (6) and cartilage (122) exhibit low synthesis of type-s p e c i f i c productsofollowed by a rapid increase and overt cytodifferentiation that i s very si m i l a r to the pancreatic sequence.tbThus, the scheme proposed by Rutter (14) may be a universal characteristic of cyt o d i f f e r e n t i a t i o n , although i n the case of ca r t i l a g e , the ubiquitous production of chond-r o i t i n sulphate makes the temporal sequence of cytodifferentiation of t h i s tissue d i f i c u l t to follow (42). The possible o r i g i n of the adrenal cort-i c a l c e l l s described here from capsular "stem" c e l l s and the implicated role of the capsular c e l l s in vivo to provide parenchyma precursor c e l l s during adrenal c o r t i c a l regeneration increases the temptation to compare f'ibroblkst-like adrenal c e l l s to embryonic c e l l s and speculate that in vitro, .cap£-: \*x ra.Ws -oy be. azize' ..d '.va pro 'jcli.vc. 133 and perhaps, in vivo, capsular c e l l s may be arrested i n a protodiff-erentiated state to be released by s p e c i f i c physiological events allowing continuation of their c y t odifferentiation i n a manner similar to embryonic development. I t i s further tempting to speculate that the presence of anionic ex t r a c e l l u l a r matrix i s responsible for holding c e l l s i n that state i n the normal adult animal. The modulation of adrenal c o r t i c a l c e l l s i n form and function as w e l l as the a b i l i t y of adrenal f i b r o b l a s t - l i k e c e l l s to respond to ACTH suggests, according to Rutter scheme of cytodifferentiation (14,17) that the adrenal c e l l s are behaving i n a manner more similar to adult than embryonic c e l l s . However, the difference i n steroid production of f i b r o b l a s t - l i k e c e l l s compared to the e p i t h e l i a l - l i k e c e l l s even i n the presence of ACTH i s more extensive than modulations of adult parenchymaasteroid production i n response to environmental parameters in vivo. F i n a l l y , the d u a l i s t i c nature of adrenal f i b r o b l a s t - l i k e c e l l s i s characteristic of a more primitive state of cytodifferentiation. The characteristics outlined above are consistent with the p o s s i b l i t y that the adrenal c e l l s described i n this report, are representative of an embryonic-like state with the e p i t h e l i a l - l i k e c e l l s representing the more advanced developmental stage compared to the f i b r o b l a s t - l i k e form. 134 I I . The co n t r o l of adrenal c o r t i c a l c e l l steroidogenesis, morphology  and movement in vitro by metachromatic e x t r a c e l l u l a r matrix (MECM). It i s becoming apparent that macromolecules of the e x t r a c e l l u l a r matrix exert an influence on c e l l c y t o d i f f e r e n t i a t i o n . Components of the e x t r a c e l l u l a r matrix influence the phenotypic.expression of chond-rocytes (51), corneal epithelium (8) and appear to possess developmentally i n s t r u c t i v e properties guiding the organogenesis of many e p i t h e l i a l organs (42). The mode of action of the c r i t i c a l molecules and even, i n the case of e p i t h e l i a l organ formation, the i d e n t i t y of the molecules i s not known. The present study implicates components of polyanionic e x t r a c e l l -u l a r matrix (MECM) i n l i m i t i n g adrenal c o r t i c a l c e l l c y t o d i f f e r e n t i a t i o n . in vitro. MECM production appears to mediate the decline o f . s t e r o i d production,, the increase i n c e l l migration and the development of a f i b r o b l a s t - l i k e morphology that occurs upon exposure of e p i t h e l i a l -l i k e c e l l s to FCS-medium. The a b i l i t y of charged growth substratum to mimic the influence of endogenous MECM suggests that i t s e f f e c t on adrenal c o r t i c a l c e l l behavior i s e x t r a c e l l u l a r and due to i t s charge. A. Control of _te_rc_id_ p_rjodu.ction._by_ MECM The mechanism(s) by which exposure to charged molecules such as matrix components, hyaluronic acid or sulphonated/polylysine treated surfaces i n h i b i t s fluorogenic s t e r o i d prodcution i s not e n t i r e l y c l e a r . I n h i b i t i o n of steroidogenesis could be caused i n d i r e c t l y by the a l t e r a t -ions i n growth patterns observed i n the present study i n response to a l l charged environmental factors except chondroitin sulphate. The possible existence of such a causal r e l a t i o n s h i p would seem Ito be suggested by the 135 fact that chondroitin sulphate was. unique among the charged molecules i n i t s lack of i n h i b i t i o n of steroidogenesis. However, since a drop i n steroid production could occur i n the absence of overt morphologic changes ( i e . when e p i t h e l i a l - l i k e c e l l s were exposed to DON i n FCS-medium or grown on brushed surfaces), i t i s probable that growth pattern a l t e r -ations are not a cause, but rather a r e s u l t , of e a r l i e r changes that precipitate the drop i n steroid production. The state of adrenal cort-i c a l c y t o d i f f e r e n t i a t i o n , i n terms of steroid production, appears to eorrelatermore consistently with an increase i n membrane undulation rather than alterations i n growth patterns since i n a l l culture condit-ions where steroid production dropped, an increase i n c e l l surface mem-brane movement.was observed. C e l l membrane movement can be expected to reduce the extent of homotypic c e l l - c e l l contact and interaction (21,22) which appears to be required for cytodifferentiation of many tissues. However, membrane movement i s a complex phenomenon and i t s stimulation undoubtedly alters s p e c i f i c arrangements as wel l as rheological prop-er t i e s of the membrane which appear to be important determinants of the a c t i v i t y of some membrane-associated enzymes (123,124). I t would appear that a major event during cytodifferentiation would be a process that confers s t a b i l i t y to membranes so as to prevent movement, i f , i n fa c t , such movement i s detrimental to maximal adult phenotypic expression .(123). In this context, the generally reported increase i n membrane f l u i d i t y (125) concurrent with malignant transformation i s interesting i n view of the often observed decrease or al t e r a t i o n -in the expression of tissue s p e c i f i c properties accompanying such transformation. It should be pointed out that the drop i n steroid production i n the presence of charged pfaefcorsrs was greater than i n the i n the absence of charged environmental factors ( i e . adrenal c e l l s exposed to DON i n 136 FCS-medium-cells grown on brushed or n i t r i c acid-treated surfaces), suggesting v either that MECM-exerts'an effect on steroid biosynthesis that i s additional to the postulated involvement of increased membrane undulation or that MECM i n h i b i t s production v i a a mechanism unrelated to i t s effect on membrane movement per se. I n h i b i t i o n could result d i r e c t l y through changes i n a c t i v i t y of membrane-associated enzymes such as adenyl cyclase whose product, c y c l i c AMP, has been shown to be an important i n t r a c e l l u l a r molecule c o n t r o l l i n g adrenal c o r t i c a l phenotype in vivo 423/) and in vitro (72-75). This speculation may be correct since charged molecules have been shown to i n h i b i t basal and hormone-stimulated adenyl cyclase act-i v i t y i n isolated membranes of thyroid c e l l membrane preparations (126). B. Cc5it_rol_of_ _;eil_mc^rp_h_ology_ by_ME_CM The development of a bipolar morphology i n FCS-medium appears to depend upon the presence of MECM since the appearance of such a form cor-.£§ItiSae4l8s8'ly*witH tK§ pWduetiion - of histochemically demonstrable, MECM and i n h i b i t i o n of i t s synthesis by DON prevented i t s development. Furthermore, growth i n HS-medium i n the presence of hyaluronic acid or on sulphonated surfaces also promoted the development of a fibrob-l a s t - l i k e morphology. That the lack of morphologic a l t e r a t i o n i n the presence of DON i n FCS-medium was due to toxic effects of this drug rather than an i n h i b i t i o n of MECM production i s unlikely since the addition of glutamine, glucosamine or glucosamine-6-phosphate reversed i t s ' effects. The a b i l i t y of charged substratum totentir'ely.lreprodueeifhe influence of MECM on c e l l form as wel l as the close apposition of sheets of endog-137 enous matrix i n FCS-medium suggest that the development of a bipolar morphology may require attachment to a charged growth surface with structural i n t e g r i t y . Such an interpretation would be i n agreement with the observations of Maroudas (68) and Harris (127) demonstrating that f i b r o b l a s t - l i k e c e l l s exert considerable tension on their growth surface and therefore require a substratum of structural i n t e g r i t y for the main-tainance of a bipolar form. Thus, hyaluronic acid i n solution or adsorbed to the growth surface could conceivably a l t e r c e l l morphology to a lesser extent than a sulphonated surface because of limited a b i l i t y to coat the growth surface, thereby presenting either a less densely charged sub-stratum or one of i n s u f f i c i e n t s t r u c t u r a l i n t e g r i t y to support a f i b -r o b l a s t - l i k e form. In contrast to hyaluronic acid, chondroitin sulphate; either may not interact with the growth surface at a l l or provide even less r i g i d i t y than hyaluronic acid. The mechanism(s) by which a charged substratum of structural integ-r i t y promotes a f i b r o b l a s t - l i k e morphologyiis not clear although the increased c e l l attachment to charged surfaces suggests that-strong.adhesion ;may foe. ;re's,p'on*sible. C. (>°ntro_ _ f _ c e l l movement_by_ MECM The environmental parameters cont-roiDljing mammalian c e l l movement in the adult and during morphogenesis are v i r t u a l l y unknown. Although recent evidence has implicated the timed production and s p a t i a l d i s t -r i bution of embryonic ex t r a c e l l u l a r matrix components, p a r t i c u l a r l y hyaluronic acid, i n controlling and directingmtgaratiiron of certain em-bryonic c e l l s in vivo (44-46), Conrad and Spooner (>106) were unable to demonstrate a correlation between c e l l movement and matrix production in vitro. The present study d e f i n i t i v e l y shows that MECM production mediates adrenal?cortical c e l l movement in vitro since DON, which i n h i b i t MECM production, prevents migration. That DON i s not exerting a toxic effect and thereby d i r e c t l y preventing c e l l movement i s suggested by the r e v e r s i b i l i t y of th i s i n h i b i t i o n upon addition of glucosamine, glucosamine-6-phosphate or glutamine concomitanti- with detectable MECM production, as w e l l as the lack of effect of DON added d i r e c t l y to actively moving f i b r o b l a s t - l i k e c e l l s that had previously synthesized MECM and which at the end of the experiment ( 36h later) s t i l l exhib-i t e d MECM. These l a s t results suggest that the i n a b i l i t y of DON to affect locomotion of 3T3 c e l l s , which produce large amounts of ECM, in Conrad and Spooner's study (106) , was due to a similar presence of MECM probably synthesized p r i o r to the addition of DON. The a b i l i t y of DON to i n h i b i t adrenal c e l l locomotion i s interesting since this drug has been shown to be teratogenic i n rat fetuses even when i t s i n h i b i t o r y effect on c e l l d i v i s i o n (104) i s , by-pp.assed* by. the t addition of adenine and guanine. Although the molecular basis for teratogenesis i s not wel l understood (12), the disruption of the reg-ulated c e l l movement during morphogenesis could w e l l lead to abnormal-i t i e s . The mechanism(s) by which MECM promotes migration i s not clear. However, since charged growth surfaces mimic thiseef•fecto.and since these surfaces also increased the rate of c e l l attachment, MECM may increase c e l l movement by increasing cell-substratumaadhes'ibn. In conclusion, evidence presented here suggests that MECM production controls the migration behavior of adrenal c o r t i c a l c e l l s i n FCS-medium,. 139 p o s s i b l y by i n c r e a s i n g c e l l - s u b s t r a t u m . a d h e s i o n . These r e s u l t s - a l s o suggest that the l a c k of movement i n HS-medium may r e s u l t from absence or very low amounts of MECM. FCS l i k e l y s t i m u l a t e s m a t r i x p r o d u c t i o n i n a v a r i e t y of c e l l types and i t s common use as a n u t r i e n t supplement may be r e s p o n s i b l e for the g e n e r a l l y observed h i g h m o t i l e r a t e s i n t i s s u e c u l t u r e . 140 CONCLUSIONS Developmental b i o l o g i s t s have long recognized that the coord-ination of c e l l d i v i s i o n , cellmmovement and c e l l u l a r interactions are prerequisite to normal development. I t i s becoming apparent that i n the embryo, components of the embryonic e x t r a c e l l u l a r matrix may mediate many of these events, at least during s p e c i f i c stages i n development. For instance, the production of the polyanion, Hyaluronic acid, has been implicated i n controlling and directing a sequence of events essential to normal chondrogenesis including promotion of morphogenetic movements, i n h i b i t i o n of overt cytodifferentiation and eventual aggreg-ation i n the correct s p a t i a l arrangement i n the embryo. Polyanionic e x t r a c e l l u l a r matrix may w e l l act as a universal "morphogen", directing a part of the d i f f e r e n t i a t i v e process of other mesodermal.tissues. Based on evidence from this study, such factors may do so not only by alt e r i n g growth patterns but by d i r e c t l y affecting, v i a charge properties, the metabolic machinery of the c e l l s . Thus MECM i n the embryo may primarily direct morphogenetic movements so that tissues can be brought into proximity with one another, but i t may also serve aeregulatory function by suppressing the production of i n t r a c e l l u l a r factors such as c y c l i c AMP, that regulate functional expression. I t may also diminish the responsive-ness to other morphogenetic environmental factors, thereby preventing premature homo- and/or heterotypic c e l l interactions leading to terminal d i f f e r e n t i a t i o n , that might disrupt the timing and sequence of the morphogenetic process necessary for f i n a l expression of the adult phenotype. The demonstration of adrenocortical characteristics i n f i b r o b l a s t - l i k e c e l l s resembling adrenal capsular tissue in vivo and the demonstration that MECM controls the degree of expression of these cha r a c t e r i s t i c s , 141 suggests a need t o . r e f o c u s a t t e n t i o n to the capsule as a p o s s i b l e stem c e l l source i n the a d u l t adrenal cortex. The a b i l i t y of MECM in vitro to i n h i b i t a d r e n a l c o r t i c a l phenotypic expression suggests that in vivo, matrix may w e l l be preventing e x p r e s s i o n of a d r e n o c o r t i c a l c h a r a c t e r i s t i c s i n capsular t i s s u e . 142 SUMMARY- -1. Previously, culture techniques for growing normal adult adrenal c o r t i c a l c e l l s were limited to primary cultures where the presence of mixed c e l l types made biochemical analysis d i f f i c u l t to interpret. 2. Culture conditions are described where apparently homogeneous secondary monolayers can be maintained as either of two morphologic and functional c e l l forms. A study was undertaken to determine the precise environmental as well as c e l l u l a r factors controlling the. ' function of adrenal c o r t i c a l c e l l s i n secondary monolayer culture and to delineate their c e l l u l a r o r i g i n within the adult adrenal cortex. 3. An e p i t h e l i a l - l i k e form was obtained when c e l l s were subcultured as intact groups from confluent primary monolayers into HS-medium. A f i b r o b l a s t - l i k e form was obtained when c e l l s were either subcultured from non-confluent primary cultures, dissociated into single c e l l s and then plated into secondary culture or grown i n medium containing f e t a l calf serum (FCS-medium). 4. Functional properties of the two morphologic forms are described including histochemistry, [4- l 4C]pregnenolone metabolism, endogenous fluorogenic production and steroidogenic response to ACTH. Adrenal e p i t h e l i a l - l i k e c e l l s are more highly cytodifferentiated than f i b r o -b l a s t - l i k e adrenal c e l l s i n terms of histochemical staining, adreno-co r t i c o i d formation from [4- 1 4C]pregnenolone and amount of fluorogenic steroid production. The low levels of steroid produced by adrenal f i b r o b l a s t - l i k e c e l l s are reminiscent of the protodifferentiated stage i n the cytodifferentiation of many embryonic tissues. 5. U l t r a s t r u c t u r a l characteristics i n the presence and absence of adrenocorticotrophic hormone are described i n both morphologic forms. Lack of mitochondrial cristae changes such as observed i n fascicular and, under certain conditions, i n glomerulosa c e l l s i n vivo and i n primary c u l t u r e suggest that secondary monolayers of adrenal c e l l s described here do not der i v e from these a d u l t parenchymal c e l l s . Rather, the u l t r a s t r u c t u r a l c h a r a c t e r i s t i c s are suggestive of e a r l y embryonic forms of adrenal c o r t i c a l c e l l s . 6. A d d i t i o n of FCS-medium to h i g h l y f u n c t i o n a l adrenal c o r t i c a l e p i t h e l i a l - l i k e c e l l s caused t h e i r modulation to a f i b r o b l a s t - l i k e form and f u n c t i o n suggesting that c u l t u r e c o n d i t i o n s ( i e . HS-medium vs FCS-medium) are not s e l e c t i n g f o r two d i s t i n c t c e l l types but, r a t h e r , r e g u l a t i n g the extent of adrenal c o r t i c a l phenotypic expression The drop i n s t e r o i d production i n the presence of FCS-medium c o r e l a t e d w i t h the development of a f i b r o b l a s t - l i k e morphology, increased meta-chromatic e x t r a c e l l u l a r matrix production (MECM), increased c e l l m i g r a t i o n and increased c e l l d i v i s i o n . An attempt was made to d e l i n -eate which of these a l t e r a t i o n s i n c e l l behavior, i f any, were r e s -p o n s i b l e f o r the observed r e g r e s s i o n i n terms of s t e r o i d production. 7. The i n f l u e n c e of MECM production was i n v e s t i g a t e d by (a) i n h i b -i t i n g i t s production i n FCS-medium w i t h 6-diazo-5-oxo-L-norleucine (DON) and (b) by adding, exogenously, components of MECM such as hya l u r o n i c a c i d to adrenal e p i t h e l i a l - l i k e c e l l s i n HS-medium. MECM production i n FCS-medium appears to be re s p o n s i b l e f o r the re g r e s s i o n of adrenal c o r t i c a l e p i t h e l i a l - l i k e c e l l s exposed to t h i s serum supp-lement s i n c e (a) DON prevented not only MECM production but the dev-elopment of a f i b r o b l a s t - l i k e morphology, c e l l m i g r a t i o n and the l a r g e drop i n s t e r o i d production and (b) conversely, a d d i t i o n of h y a l u r o n i c a c i d promoted a b i p o l a r morphologic form and p r e c i p i t a t e d a l a r g e drop i n s t e r o i d production. 8. Growth on charged a r t i f i c i a l s u b s t r a t a mimicked the effetotst of endogenously produced and exogenously added MECM suggesting that the e f f e c t of MECM on adrenal c o r t i c a l c e l l i n v i t r o i s e x t r a c e l l -u l a r and due to charge. 9. The p o s s i b l e mechanism(s) by which e x t r a c e l l u l a r charged f a c t o r s i n h i b i t s t e r o i d production and concomitantiyy' promote m i g r a t i o n and assumption of a f i b r o b l a s t - l i k e morphology are discussed. A r o l e of MECM i n the a d u l t adrenal cortex and during the morphogenesis of t h i s gland i s p o s t u l a t e d . 144 LITERATURE CITED 1. Schubert, D. and F. Jacob. 1970. 5-Bromodeoxy-uridine-induced d i f f e r e n t i a t i o n of a neuroblastoma. Proc. N a t l . Acad S c i . U.S.A. 67: 247-254. 2. Prasad, K. 1971. Transmission of mouse neuroblastoma by a c e l l -f r e e e x t r a c t . Nature. 228: 997-999. 3. Furmanski, P., D.Schuman and M. Lub'in. 1971. 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Functional and morphological observations on rat adrenal zona.glomerulosa c e l l s i n culture. Endocrinol. 95: 1240-1251. 114. Roos, T.B. 1967. Steroid synthesis in.embryonic and f e t a l rat adrenal t i s s u e . Endocrinol. 81: 716-728. 115. B r e s s l e r , R.S. 1973. Myoid c e l l s i n the capsule of the adrenal gland i n monolayers derived from cultured adrenal capsules. Anat. Rec. 77: 525-531. 116. Nickerson, P.A., A.C. Brownie, F.R. Skelton. 1969. An electron microscopic study of regenerating adrenal gland during the development of adrenal regeneration hypertension. Amer. J. Pathol. 57: 335-364. 117. Baker, D.D. and R.N. B a i l i f f . 1935. Role of capsule i n supra-renal regeneration. Proc. Soc. Exp. B i o l . Med. 40: 117-132. 118. Ingle, D.J. and G.M. Higgens. 1935. Autotransplantation and reg-eneration of the adrenal gland. Endocrinol. 22: 458-464. 155 119. Sabatini, D.D. , H.B. Bleichmar and E.D.P. de Robertis. 1963. Ul t r a s t r u c t u r e of the regenerating adrenal cortex of the r a t . Acta Endocrinol. 34(suppl. 51): 453, #227. 120. Schaberg, A. 1955. Regeneration of the adrenal cortex in vitro. Anat. Rec. 122: 205-222. 121. Penney, D.P. , D.I. Patt and W.C Dixon. 1963. The f i n e structure of regenerating, adrenal c o r t i c a l autotransplants i n the rat. Anat. Rec. 146: 319-335. 122. K v i s t , T.N. and C.V. Finnegan. 1970. The d i s t r i b u t i o n , of glycos-aminoglycans i n the a x i a l region of the developing chick embryo. I I . Biochemical analysis. J . Exp. Zool. 175: 241-258. 123. Edelman, G.M. 1976. Surface modulation.in c e l l recognition and c e l l growth. Science. 192: 218-226. 124. Barnett, R.E., L.T.. Furcht and R.E. Scott. 1974. Differences i n membrane f l u i d i t y and structure i n contact i n h i b i t e d and transformed c e l l s . Proc. Nat. Acad. S c i . U.S.A. 172: 1992-1994. 125. Sachs, L. 1975. Lectins as probes f o r changes i n membrane dyn-amics i n malignancy, a n d . c e l l d i f f e r e n t i a t i o n . i n Dynamics of c e l l surface membranes. 127-139. 126. Wolff, J. and G.H. Cook. 1975. Charge e f f e c t s i n the a c t i v a t i o n of adenylate cyclase. J . B i o l . Chem. 250: 6897-6903. 127. H a r r i s , A.K. 1973. C e l l surface movements rela t e d to c e l l loco-motion, i n Ciba Found. Symp. 14: 3-26. Appendix 1. Procedure for A5-3B-hydroxysteroid dehydrogenase (Levy et al. 1959) 156 Constituent Amount F i n a l Molal 1. DHEA1 0.2 mg ImM ' 2. Methyl formamide 2 0.5 ml IM 3. Nitro BT s o l u t i o n 3 1 mg/ml of H 20 1.0 0.16mM 4. Nicotinamide s o l u t i o n 4 1.6 mg/ml H 20 0.7 ml 4mM 5. NADP s o l u t i o n 5 3 mg/ml H 20 0.8 ml 0.54 M 6. Phosphate b u f f e r 6 0.1M pH 7.1-7.4 4.0 ml 0.057mM 1 Purchased from Steroloids Inc., Pawling, N.Y. 2 Fisher S c i e n t i f i c Co. Ltd., Chem. Man. Div. Fairlawn, N. Jersey,'U.S.A. 3 Kindly donated by Drs. Burton and Richards, Cancer Research I n s t i t u t e , U.B.C., Vancouver, B.C. 4 Fisher S c i e n t i f i c Co. Ltd., Chem. Man. Div. Fairlawn, N. Jersey, U.S.A. 5 Kindly donated by Drs. Burton and Richards, Cancer Research I n s t i t u t e , U . B. C., Vancouver, B ,'C. 6 Fischer S c i e n t i f i c Co. Monolayers were not fi x e d with glutaraldehyde as reported previously (Levy et al, 1959, Kahri, 1966) but were r a p i d l y frozen by exposure to dry i c e and then allowed to return to room temperature. This procedure was repeated several times to ensure membrane rupture. Monolayers were then immediately stained with above s o l u t i o n , or stored at -20 C u n t i l needed. Monolayers were incubated i n the above mixture at 37 C for 15-30 minutes or u n t i l formazan ' deposition was observed. Control monolayers were incubated i n i d e n t i c a l manner but with a so l u t i o n lacking DHEA. 157 Appendix I I . Chromatographic Behaviour of Radiometabolites Produced by Adrenal Cultures and Muscle Fascia Fibroblasts (Rfc = testosterone) Radiometabolites PPC 1 (R_) PPC 11 (R t) TLC 1 (Rf) TLC 1A (Rf) TLC 11 (Rf) A) Adrenal E p i t h e l i a l Cultures Band 1 } o r i g i n 0.17 0.29 0.53, 0.61 0. 79 2. 0.33 0.55 0.45 0.60 0.82 3. 1.78 R.O.2 0.7.1 0.88, 0.79 0.63 0.80 4. 2.39 R.O. 0.76 0.71 0.76 5. 1.17 R.O. 0.83 0.83, 0.75 0.68, -origin 0.95 B) Adrenal Fi b r o b l a s t i c Cultures Band 1. o r i g i n 0.38, o r i g i n - - -2. 0.33 0.55 0.45 0.60 0.82 3.1 1.78 R.O. 0.71 0.88, 0.79, 0.63 0.80 4.1 2.39 R.O. 0.76 > 0.71 0.76 5. 1.17 R.O. 0.83 • 0.83, 0.75, 0.68, o r i g i n 0.95 C) Muscle Fascia Fibroblasts Band 1. 2. o r i g i n 1.17 0.38, o r i g i n 0.44 R.O. 0.83 0.83, 0.75 0.95 Radiometabolites produced i n response to ACTH (lOOmU/ml medium/day). Metabolites ran off with the solvent front. (cont'd.) Appendix I I (Continued) PPC 1 - Whatman No. 1 paper impregnated with MeOH:propylene glycol (1:1, V:V) and run i n l i g r o i n for 24 h. PPC 11 - Whatman No. 1 paper impregnated with MeOH:propylene gly c o l (1:1, V:V) and run i n benzene:hexane. TLC 1 - S i l i c a Gel G plates (Eastman Kodak) run i n Benzene:Ethylacetate (3:1, V:V). TLC 1A - Radiometabolites acetylated (See Appendix IV) and run i n TLC-1. TLC 11 - S i l i c a Gel G plates run i n chloroform:ethylacetate (1:50, V:V). 158 159 Appendix I I I . Chromatographic Procedures A. V i s u a l i z a t i o n of A' Steroids a. p_hc_sp_homo_l£bd_a_te 1. dissolve 8 g of phosphomolybdic acid i n 100 ml ethanol. 2. spray onto paper or th i n layer. 3. heat at 55°C u n t i l dry and steroid shows as a dark area. b. Zimmerman reaction 1. dissolve 2g of dinitrobenzene i n 100 ml ethanol. 2. soak paper or TLC i n t h i s solution. 3. soak paper or TLC i n 2.5N KOH (lOg i n 100 ml ethanol). 4. steroid w i l l show as pink spot. B. Acetylation and hydroxylation of steroids with free hydroxyl groups 1. acetylate compounds by adding to sample (lOOug), 0.1 ml of pyridine and 0.1 ml of acetic ^anhydride. 2. heat mixture at 57°C for 2 h or leave at room temperature for approx-imately 12 h. 3. wash with absolute methanol twice. 4. hydroxylate compounds by adding 0.3 ml methanol and 1.0 ml of 0.1N NAOH i n 70% methanol. 5. gas with ^  approximately 1 min to provide environment conducive- to reduction. 6. stopper and wrap with s i l v e r f o i l and paper. Store for 12h in dark plac 7. Extract once with chloroform and discard the aqueous layer. 8. wash once with d i l u t e acetic acid to neutralize remaining NAOH. 9. wash once with d i s t i l l e d water to remove acetic acid. APPENDIX IV 160 Preparation of c e l l s for transmission electron microscopy A. Dehydration and embedding 1. 50% ETOH, 5 minutes 2. 20% ETOH, 5 minutes 3. 90% ETOH, 5 minutes 4. 100% ETOH, 5 minutes 5. 100% ETOH, 5 minutes 6. 100% ETOH, 5 minutes 7. 100% ETOH: propylene oxide (1:), 10 minutes 8. propylene oxide, 10 minutes 9. propylene oxide, 10 minutes 10. propylene oxide: Epon (1:1), 1 hour M l . propylene oxide: Epon (1:3), 1 hour 12. Epon, 1 hour 13. Fresh Epon, 24 hours at 37°C, then overnight at 56oC. B. Epon s o l u t i o n Stock s o l u t i o n A: Epon 812 Resin 62 ml Dddecenyl Succinyl Anhydride (DDSA) 100 ml Stock s o l u t i o n B: Epon 812 Resin 100 ml Nadic Methyl Anhydride (NMA) 89 ml Working s o l u t i o n : Mix s o l u t i o n A and B (2:3), then add 2% by volume, the accelerator dimethylamino-methyl phenol (DMP-30-Tris) 161 Appendix V: . Calculations for Determining Charge Density of Sulphonated P e t r i Dishes and Toluidine Blue''' Standard Curve. ml Dye mg Toluidine Optical mg per-.:Unit Solution Blue Density Optical Density 0.01 0.0001 0.004 0.025 0.05 0.005 0.014 . 0.035 0.1 0.001 0.026 0.038 0.2 0.002 0.05 0.040 0.3 0.003 0.072 0.041 0.4 0.004 0.088 0.045 0.5 0.005 0.120 0.041 0.041 Average 1 Toluidine Blue, 2% stock solution, pH 2.5 was diluted 1000 times p r i o r to taking samples for reading on a Junior Coleman colorimeter. There are 282 grams i n 1 mole of Toluidine Blue or 6 x l 0 2 3 molecules (Avagadros number). Therefore . Ig=2.1xl0 2 1 molecules and lmg=2.1xl0 1 8 molecules per p e t r i dish which has approximately a surface area of 1137 mm2(1J2, = 2__, 11=17.5) 7-=1.8xl0 1 5 dye molecules bound/mm2 since lR = meters = 10- 7 mm, 1^2=10"1 Vn 2 X so that i n 100A°2, there would t h e o r e t i c a l l y be 1.8 x 10 3 molecules of bound dye. example: i f o p t i c a l density of a treated surface i s .088, then from standard curve we have .041 x .088 = .0036 ug but to adjust for the d i l u t i o n factor (see Table ), we multiply by 30 to get .108 mg dye bound to p e t r i dish surface. since lmg=1.8 x 10 5 molecules per mm2, .108 mg = .195 x 10 1 5 molecules/ mm2 = 1.95/A52 or 195/100F. PUBLICATIONS Slavin s k i , E.A., J.W. J u l l and N. Auersperg. rat adrenal glands. In V i t r o . 7:258-259. 1972. Culture of c e l l s from normal Slavi n s k i , E.A., N. Auersperg and J.W. J u l l . 1973. c e l l s i n v i t r o . Proc. Can. Fed. B i o l . S c i . 16: 58. Di f f e r e n t i a t i o n of adrenal Sl a v i n s k i , E.A., N. Auersperg and J.W. J u l l . 1974. Propagation i n v i t r o of functional rat adrenal c o r t i c a l c e l l s : modulation of the differentiated state by culture conditions. In V i t r o , 9: 260-269. Sla v i n s k i , E.A., J.W. J u l l and N. Auersperg. 1976. Steroidogenic pathways and trophic response of cultured adrenocortical c e l l s i n different states of d i f f -erentiation. J. Endocrin. 69: 383-393. Slavi n s k i , E.A. and N. Auersperg. 1976. Trophic response to ACTH of cultured normal adult rat adrenal c o r t i c a l c e l l s i n different states of d i f f e r e n t i a t i o n . Proc. Can. Fed. B i o l . S c i . 19: 92. Slavinski, E.A. and N. Auersperg. 1976. The role of ext r a c e l l u l a r matrix i n controlling the phenotypic expression of normal adult rat adrenal c o r t i c a l c e l l s i n v i t r o . J. C e l l . B i o l , i n press. 

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