CULTURED ADRENOCORTICAL CELLS IN VARIOUS STATES OF DIFFERENTIATION: ELECTRON MICROSCOPIC

CHARACTERIZATION AND ULTRASTRUCTURAL RESPONSE TO ADRENOCORTICOTROPHIN E. A. SLAVINSKI-TURLEY AND N. AUERSPERG Cancer Research Centre and Department of Zoology, University of British Columbia, Vancouver, B.C. V6T1W5, Canada

(Revised manuscript received 25 January 1978) SUMMARY

The ultrastructure and response to ACTH of subcultured rat adrenocortical cells in two morphological and functional states are described. Fibroblastic cortical cells, which produce low levels of corticosterone, resembled myoid cells from the adrenal capsule : they formed fibrous extracellular matrix and basement membranes and contained dilated rough endoplasmic reticulum (RER), cytofilaments resembling those of smooth muscle and lamellar mitochondrial cristae. Stimulation with ACTH for 3 days increased steroid production from 0\m=.\01 to 0\m=.\56\g=m\g106 cells\m=-\124 h\m=-\1, increased the amount of smooth endoplasmic reticulum (SER) and greatly reduced the amounts of RER, cytofilaments, basement membranes and extracellular matrix, but did not change the mitochondrial structure. Different culture conditions produced epithelial cells which secreted high levels of corticosterone, lacked extracellular matrix, basement membranes and cytofilament accumulations but contained large lipid inclusions, SER and many mitochondria with lamellar or tubulolamellar cristae and electron-dense mitochondrial matrix bodies. Stimulation with ACTH for 3 days caused an increase in steroid production from 2\m=.\3to 30\m=.\4\g=m\g106 cells\m=-\124 h\m=-\1, an increase in the number of Golgi complexes and the amount of SER as well as a reduction in the number of mitochondrial matrix bodies and lipid inclusions. However, no ultrastructural change occurred in the mitochondrial cristae. In both forms of cell, ACTH induced a transient increase in gap junctions. These and previous results suggest that subcultured adrenocortical cells in the fibroblastic form represent stem cells, possibly originating from the capsule, whose level of differentiation can be increased by ACTH as well as by specific culture conditions. INTRODUCTION

There is much evidence to indicate that zonae fasciculata and glomerulosa cells survive in a functionally and ultrastructurally differentiated state in primary tissue culture (Kahri, 1966; Armato & Nussdorfer, 1972; O'Hare & Neville, 1973; Milner, 1975). These highly differentiated cells appear as cohesive, monolayered epithelial islands, often surrounded by fibroblastic cells. After subculture into media containing foetal calf serum, the original epithelial islands disappear and are replaced by fibroblastic cells which are often considered to represent non-steroidogenic connective tissue. However, we recently reported that these * Present address : Mergenthaler Laboratory for Biology, The Johns Hopkins University, Baltimore, Maryland 21218, U.S.A.

subcultured fibroblastic cells were adrenocortical in nature, because they synthesized small amounts of corticosterone and responded to adrenocorticotrophin (ACTH) by a change in cell shape, by conversion of [4-14C]pregnenolone to deoxycorticosterone, and by a 25-fold increase in fluorogenic steroid production. The main fluorogenic steroid secreted was corticosterone as indicated by Chromatographie analysis (Slavinski, Ml & Auersperg, 1976). That the fibroblast-shaped cells were adrenocortical rather than of a connective tissue type was further supported by the histochemical demonstration of specific dehydro¬ genase activity (Slavinski, Auersperg & lull, 1974). By modifying culture conditions, cells in secondary culture could be grown at a higher level of adrenocortical differentiation, as indicated by an epithelial morphology, lipid accumulation and production of corticosterone in amounts comparable to those produced by fasciculata cells in vitro (O'Hare & Neville, 1973; Slavinski et al. 1976). The cells assumed this epithelial form only if they were derived from confluent primary cultures that had been dissociated mechanically rather than enzymically and if the culture medium was supplemented with horse serum rather than with foetal calf serum (Slavinski et al. 1974). Exposure of the epithelial cells to foetal calf serum resulted in a rapid reversion to the fibroblastic phenotype. Cinemicrography and the timing of this reversion showed it to be due to modulation of the cells from one form to the other (Slavinski & Auersperg, 1976), indicating that the secondary fibroblastic and epithelium-like cultures represent adreno¬ cortical cells of the same lineage in different states of differentiation rather than separate cell types selected by different culture conditions. Although the adrenal cells in the epithelial form were morphologically and functionally more highly differentiated than the fibroblastic form, they differed from the original epi¬ thelial islands in primary culture in terms of growth patterns and stability of the differentiated state: they formed multilayered colonies rather than monolayers, contained large lipid inclusions and assumed a fibroblastic form under culture conditions where the epithelial phenotype of the original parenchymal cells was maintained (PI. 1, fig. 1; Slavinski et al. 197 ; Slavinski & Auersperg, 1976). It was of interest to examine the ultrastructural response of both epithelial and fibro¬ blastic forms of cortical cells in secondary culture to ACTH to determine if either form resembled glomerular or fascicular cells and if the difference in steroid production between the two forms was related to a difference in ultrastructural response. Evidence is also presented which suggests that the subcultured cells originate from a stem cell source, possibly from the capsule rather than from the parenchyma of the adrenal cortex, and that they represent less differentiated forms of cortical cells than fascicular or glomerular cells in primary culture. MATERIALS AND METHODS

Cell culture cells cultures of adrenocortical were grown from expiants of glands of 2-3 month Primary old male Fischer 334 rats, in Waymouth's medium MB 752/1 containing 25% foetal calf serum and 100 mu. penicillin and 100 µg streptomycin/ml. Secondary cultures of partially differentiated fibroblastic adrenocortical cells or more highly differentiated epithelial-like cortical cells were prepared as described previously (Slavinski et al. 191 A). Briefly, fibro¬ blastic cultures were prepared by dissociating primary outgrowths with 0-12% trypsin/Mg2+, Ca2+ free Hanks' Balanced Salt Solution and subsequently growing cells in Waymouth's medium MB 752/1 containing the antibiotics and 10-25% foetal calf serum. Epithelial cultures producing high levels of steroid were prepared by mechanically dissociating confluent primary outgrowths with a rubber-tipped glass rod and scalpel and plating the resulting cell fragments into Waymouth's medium MB 752/1 supplemented with the anti¬ biotics and 3-10% horse serum.

Incubation with ACTH Porcine ACTH (88 i.u./mg, Sigma Chemicals, St Louis, Missouri, U.S.A.) dissolved in 0-9% NaCl was added to the culture medium of the secondary cultures at a final concen¬ tration of 100 mu./ml for 1, 3 or 5 days before the concentrations of steroids were deter¬ mined and the cells were fixed for electron microscopy. Fresh ACTH was added daily for the required period of time. Steroid production Production of fluorogenic steroids by the secondary cultures was examined before prepa¬ ration for electron microscopy to demonstrate that the cultures examined in the present study were functionally comparable to those used previously for biochemical analysis (Slavinski et al. 1976). Medium was collected for 24 h (i.e. the last day of incubation with ACTH), extracted with dichloromethane and reacted with ethanolic sulphuric acid (65%, v/v) as described previously. The fluorescence of the sulphuric acid layer was determined with an Aminco-Bowman spectrophotofluorometer using an excitation wavelength of 470 nm and an emission wavelength of 525 nm.

Electron microscopy Cells in secondary culture were fixed in situ on the plastic culture surface with 2-5% glutaraldehyde in phosphate buffer, post-stained in 1% osmium tetroxide and dehydrated in a graded ethanol series. Colonies of cells were then lifted from the substratum with a sharp-edged piece of plastic and fragments were further dehydrated with propylene oxide and embedded in Epon 812 resin. Sections were stained with uranyl acetate and lead citrate and examined in a Zeiss EM 10 electron microscope. RESULTS

Fluorogenic steroid production The amounts of steroid produced by the subcultured cells in the present study were com¬ parable to those reported previously. The basal production by the fibroblastic cells was 0-01 µ^ IO6 cells-124 h-1 and rose to 0-05, 0-56 and 0-08 µg respectively after 1, 3 and 5 days stimulation with ACTH. The basal production by the epithelial cells was 2-30 µg 106 cells-1 24 h-1 and rose to 12-0, 30-4 and 3-5 µg respectively after 1, 3 and 5 days of ACTH stimulation. Previous Chromatographie analysis had shown that corticosterone is the major fluorogenic steroid released into the culture medium by both the fibroblastic and the epithelial cells and that production of this steroid reaches its peak after 3 days of treatment with ACTH (Slavinski et al. 1976).

Morphology and ultrastructure Morphological changes induced by ACTH in secondary cultures and detectable by phase optics have been described previously (Slavinski et al. 1976) and are therefore summarized here only briefly. Fibroblastic cells When viewed by phase-contrast optics, the fibroblastic cells were bipolar, multilayered and oriented in parallel array (PI. 1, fig. lo). Ultrastructurally, they resembled connective tissue fibroblasts in that they were widely separated from one another, contained extensive dilated rough endoplasmic reticulum (RER) and few mitochondria with lamellar cristae (PI. 1, fig. 2; PI. 2, fig. 4). However, unlike connective tissue fibroblasts, the cells were surrounded by distinct basement membranes, as well as by large amounts of amorphous and fibrillar extracellular matrix (PI. 1, fig. 2; PI. 2, fig. 3). The cells contained prominent accumulations of cytofilaments which ran parallel with the long axis of the cells in sections cut vertically

the growth surface, but perpendicular to plasma membranes and continuous with extra¬ cellular spaces in sections cut parallel with the growth surface (PI. 2, fig. 4). Filament bundles showed focal thickenings characteristic of smooth muscle (PI. 2, fig. 5) (Ross, 1971). The cells also contained many lipid inclusions and glycogen aggregations (PI. 1, fig. 2). Focal intercellular junctions with a gap of 7-9 nm between the plasma membranes were observed. In some sections, the central lamina of these junctions appeared to consist of electron-dense particles, spaced at a distance of about 8 nm (PI. 3, fig. 6). After treatment with ACTH for 1 day, the culture morphology of fibroblastic cells remained similar to the controls. Ultrastructurally, there was an increase in the frequency of gap junctions (PI. 3, fig. 6) but no other alterations were observed, although steroid production had increased fivefold by this time. After treatment with ACTH for 3 days, i.e. at the time of maximum steroid production, the cells were more rounded and epithelial-like when observed by phasecontrast optics. Ultrastructurally, the frequency of gap junctions had again decreased, basement membranes and extracellular material had disappeared and there was a marked reduction in the number of cytofilaments. The amount of smooth endoplasmic reticulum (SER) and the number of Golgi complexes increased (PI. 3, fig. 7), but the structure of the mitochondrial cristae remained lamellar. There were no further ultrastructural changes after treatment with ACTH for 5 days. to

Epithelial cells When viewed by phase-contrast optics, epithelial cells in secondary culture contained numer¬ ous large lipid inclusions and were cohesive and more symmetrical than fibroblastic cells (PI. 1, fig. lc). The cells with the largest lipid inclusions tended to occur in the centre of the epithelial islands. Ultrastructurally, these cells had few Golgi complexes, little RER and SER and many mitochondria. Most of the cytoplasm was taken up by lipid which often showed a crystalline staining pattern. These cells were closely apposed to one another over extensive areas, but separated by focal widenings. Numerous pinocytotic vesicles were observed at the cell surfaces (PI. 4, fig. 8). In cells with smaller lipid inclusions, RER was almost entirely absent, SER was abundant and Golgi complexes were common (PI. 4, fig. 9). Lysosomes were common and, very occasionally, contained inclusions possibly representing spaces that had been occupied by cholesterol crystals before fixation (PI. 4, fig. 10; Rhodin, 1971). These cells were less closely adherent and were sometimes connected by gap junctions resembling those in fibroblastic cultures, but present in greater numbers. In general, mito¬ chondria were more numerous in epithelial than in fibroblastic cells. Most mitochondria contained lamellar cristae in a moderately electron-lucent, granular matrix, though some had tubulolamellar cristae in a more electron-dense matrix (PI. 4, fig. 9). Mitochondrial electron-dense matrix bodies, indicative of calcium deposits (Suyama, Long & Ramachan¬ dran, 1977), were present in most of the epithelial cells examined and were particularly numerous in cells with large lipid inclusions (PI. 4, fig. 8; PI. 5, fig. 11). Similar matrix bodies

in fibroblastic cells. In further contrast to fibroblastic cells, the RER was not as dilated, there were no basement membranes, no extracellular material, no glycogen inclusions and very few cytofilaments. After treatment with ACTH for 1 day, the epithelial cells had retracted and fewer lipid inclusions were observed. As in fibroblastic cultures, there was a transient increase in the number of gap junctions, usually at the tips of adjoining cell processes. Pinocytotic vesicles decreased in number while the number of polysomes increased (PI. 5, fig. 12). After treatment with ACTH for 3 days, i.e. at the time of the maximum steroidogenic response, the number of lipid inclusions was greatly reduced and the cells appeared more rounded than before. There was a large increase in the amount of SER and the number of Golgi complexes (PI. 5, fig. 13). After treatment with ACTH for 5 days, the cells had rounded even more and retracted from the growth surface. Ultrastructurally, they resembled cells exposed to ACTH for 3 days and, in addition, contained fewer polysomes and membrane-associated ribosomes (PI. 6, fig. 14). In response to ACTH, the number of were rare

dense mitochondrial matrix bodies was greatly reduced, but there was no obvious change in the number of mitochondria and in the structure of mitochondrial cristae. The principal differences induced in the subcultured adrenocortical cells by culture conditions and ACTH are summarized in Table 1. Possibly the most striking observation illustrated by this Table is the similarity in the first five characteristics listed between ACTH-treated fibroblastic cells and epithelial control cells. The changes induced in these cellular characteristics are all in keeping with the differentiation or modulation of a myoid cell to a secretory cell, and are shown here to occur in response to trophic hormone and also in response to specific environmental conditions. Table 1.

Effects of culture conditions and ACTH* on the ultrastructure and fluorogenic steroid production of rat adrenocortical cells Fibroblastic Control

Extracellular matrix Basement membrane

SER§, Golgi complexes

Glycogen aggregations Cytofilaments Gap junctions Lipid inclusions Mitochondria Number Matrix granules Cristae

+ + + + + +

++ ++

in

Epithelial celisi

cellsf ACTH 0 0 ++

+ ++

secondary culture

0 +

Transient increase after 1 day of ACTH

ACTH

Control 0 0 ++ 0 + ++

0 0 +++

0 + Transient increase after 1 day of ACTH

+++ ++ +

++ H-

Lamellar

Lamellar

+++ ++ +

Lamellar and tubulolamellar

+ ++ +

Lamellar and tubulolamellar

Fluorogenic steroid production 001 2-30 30-40 0-56 (y.g 10e cells-1 24 h-1) 0, absent; +, few; + +, moderate amount; + + +, large amount. § SER, smooth endoplasmic reticulum. * ACTH was administered at a dose of 100 mu./ml culture medium, daily for 3 days. t Subcultured from primary culture by dissociation with trypsin; secondary culture in medium with 25% foetal calf serum. X Subcultured from primary culture by mechanical dissociation; secondary culture in medium with horse serum.

3%

DISCUSSION

In the absence of ACTH, the ultrastructure of fibroblastic adrenocortical cells in secondary culture closely resembled that of myoid cells grown from the adrenal capsule (Bressler, 1973) and from peritubular testicular tissue (de Kretser, Catt & Dufau, 1971). Like the myoid cells in testicular cultures (Dufau, de Kretser & Hudson, 1971), the secondary adrenal cortical cells are capable of synthesizing steroids as demonstrated previously by metabolism of [4-14C]pregnenolone, by endogenous production of corticosterone and by quantitative and qualitative changes in steroid metabolism in response to ACTH (Slavinski et al. 1974, 1976). Their steroidogenic capacity and changes in shape and ultrastructure in response to ACTH suggest that the subcultured fibroblastic cells are adrenocortical stem cells, possibly of capsular origin (Baker & Bailiff, 1935). This concept is supported by the histochemical demonstration of specific dehydrogenase activity (Slavinski et al. 1974) and is in keeping with the demonstration by others of fluorogenic steroid production in fibro¬ blastic cultures from the adrenal capsule (Ramachandran & Suyama, 1975).

In the absence of

ACTH, the subcultured epithelial cells ultrastructurally resembled & Neville, 1974) or fascicular cells (Kahri, 1966; Armato & O'Hare & Nussdorfer, 1972; Neville, 1973; Milner, 1975) in primary culture since they contained little SER or RER, few Golgi complexes and numerous mitochondria with electron-dense matrix granules (Suyama et al. 1977). However, mitochondrial cristae were mainly lamellar, although a variable proportion of cells did show tubulolamellar cristae,

glomerular (Hornsby, O'Hare

in keeping with reports of fascicular cells in primary culture (Milner, 1975). The subcul¬ tured epithelial cells also differed from primary cultures of cortical parenchyma in shape, growth pattern and lipid content, and in their capacity to modulate to the fibroblastic form upon exposure to foetal calf serum (Slavinski & Auersperg, 1976). The fact that the advanced state of secretory differentiation of the epithelial adrenal cells can revert to a more primitive form by a change in serum supplement distinguishes them from the morphologically more stable adrenocortical parenchymal cells in primary cultures and suggests that the epithelial cells in our secondary culture system are highly, but not irreversibly differentiated. Both forms of subcultured adrenocortical cells described here clearly exhibited ultrastructural modifications in the presence of ACTH: trophic hormone increased the formation of specialized junctions, the amount of SER and the number of Golgi complexes. In addi¬ tion, ACTH caused the loss of myoid characteristics in the fibroblastic cells and rendered them ultrastructurally similar to the epithelial form. Neither cells showed changes in mitochondrial cristae although steroid production increased, particularly in the epithelial cultures where it became comparable, on a per cell basis, to that of fascicular parenchyma in vivo (Shima & Pincus, 1969; Gill, 1972) and in primary culture (O'Hare & Neville, 1973). Based on these observations, several conclusions may be drawn. First, the ultrastructural characteristics, the lack of changes in the mitochondrial cristae and the low level of steroid production by the fibroblastic cells suggest that these cells do not originate from cortical parenchyma but rather from the capsule. Their resemblance to cultured adrenal capsular cells (Bressler, 1973) in conjunction with their steroidogenic capacity and modu¬ lation from a myoid to a secretory type of cell in response to ACTH lend strong support to the hypothesis (Baker & Bailiff, 1935) that capsular fibroblasts represent adrenocortical stem cells. Secondly, the ultrastructural resemblance to epithelial cells induced in the fibroblastic cells by ACTH, and the modulation of the epithelial cells to the fibroblastic form in response to foetal calf serum (Slavinski & Auersperg, 1976), indicate that the two forms of cells are interconvertible. They therefore appear to represent adrenocortical cells expressing different states of differentiation in response to factors in the environment, rather than separate cell types selected through variations in culture conditions. Such capacity for modulation is not unexpected in a mesodermal gland where stroma and paren¬ chyma are of common embryonic origin. Thirdly, contrary to a previous hypothesis (Kahri, 1971), alterations in mitochondrial cristae to a vesicular form do not appear to be required for the large increases in ACTH-mediated production of corticosterone. Maximum corti¬ costerone production was correlated more closely with an increase in the amount of SER and the number of Golgi complexes, a result which agrees with the observations of Stark, Gyevai, Bukulya, Szabo, Szalay & Mihaly (1975) on the developing hormone responsiveness in human and cat foetal adrenal glands. Finally, the differences in steroid production between fibroblastic and epithelial forms of subcultured adrenocortical cells cannot be ascribed to qualitative differences in the ultrastructure of organelles involved in steroid biosynthesis in the presence or absence of ACTH. Since the epithelial cells contained more mitochondria, lipid and SER than the fibroblastic cells, their high capacity for corticosterone production may reflect the greater quantities of these organelles. The stimulation of gap junction formation in adrenocortical cells exposed to ACTH has been noticed previously (Armato & Nussdorfer, 1972) but not emphasized. Such stimulation is interesting since a similar phenomenon has been observed in granulosa cells in response to trophic hormones (Albertini, Fawcett & Olds, 1975) as well as in developing

tissues (Trelstad, Hay & Revel, 1967; Benedetti, Dunca & Blocmendal, 1974). Since nucleotides are able to cross gap junctions (Pederson, Sheridan & lohnson, 1976), it has been suggested that these junctions maximize and synchronize the responses to trophic

hormones. The width of the gap between adjoining plasma membranes in the junctions observed in this study was 8-9 nm, a value which exceeds that most frequently found in gap junctions (2-4 nm). This difference might be the result of physiological variation or varia¬ tions in fixation methods. Alternatively, the junctions observed in this study might be similar to the wider septate-like junctions which have been described as characteristic of steroid-secreting and, particularly, adrenocortical tissues (Friend & Gilula, 1972). It was suggested that the function of these wider junctions might be the formation of microchannels between cells for efficient distribution of secretory products. A more detailed analysis is required for the precise identification of the junctions observed in the present study. We are grateful to Mr K. S. Wong for expert technical assistance. This work was supported by a grant and a Research Associateship to N. Auersperg from the National Cancer Institute of Canada. REFERENCES

Albertini, D. F., Fawcett, D. W. & Olds, P. J. (1975). Morphological variations in gap junctions of ovarian granulosa cells. Tissue and Cell 3, 389-405. Armato, U. & Nussdorfer, G. G. (1972). Tissue culture of adult rat decapsulated adrenal glands. A metho¬ dological, ultrastructural and morphometric investigation. Zeitschriftfür Zellforschung und mikroskopische Anatomie 135, 245-273. Baker, D. D. & Bailiff, R. N. (1935). Role of capsule in suprarenal regeneration. Proceedings of the Society for Experimental Biology and Medicine 40, 117-132. Benedetti, E. L., Dunca, I. & Blocmendal, H. (1974). Development of junctions during differentiation of lens fibers. Proceedings of the National Academy of Sciences of the U.S.A. 71, 5073-5077. Bressler, R. S. (1973). Myoid cells in the capsule of the adrenal gland and in monolayers derived from cultured adrenal capsules. Anatomical Record 77, 525-531. Dufau, M. C, de Kretser, D. M. & Hudson, B. (1971). Steroid metabolism by isolated rat seminiferous tubules in tissue culture. Endocrinology 88, 825-832. Friend, D. S. & Gilula, N. B. (1972). A distinctive cell contact in the rat adrenal cortex. Journal of Cell Biology 58, 148-163. Gill, G. W. (1972). Mechanism of ACTH action. Metabolism 21, 571-588. Hornsby, P. J., O'Hare, M. J. & Neville, A. M. (1974). Functional and morphological observations on rat adrenal zona glomerulosa cells in culture. Endocrinology 95, 1240-1251. Kahri, A. I. (1966). Tissue culture of the adrenals. Acta Endocrinologica 52, Suppl. 108, 1-96. Kahri, A. I. (1971). Inhibition by cycloheximide of ACTH-induced internal differentiation of mitochondria in cortical cells in tissue culture of fetal rat adrenals. Anatomical Record 171, 53-80. de Kretser, D. M., Catt, K. J. & Dufau, M. L. (1971). Studies on rat testicular cells in tissue culture. Journal of Reproduction and Fertility 24, 311-318. Milner, A. J. (1975). An ultrastructural approach to the study of endocrine cells in vitro. Methods in Enzymology 39, Part D, 157-165. O'Hare, M. J. & Neville, A. M. (1973). Morphological response to corticotrophin and cyclic AMP by adult rat adrenocortical cells in monolayer culture. Journal of Endocrinology 56, 529-536. Pederson, D. C, Sheridan, J. D. & Johnson, R. G. (1976). Evidence for nucleotide transfer during junctional formation in culture. Journal of Cell Biology 70, 338a. Ramachandran, J. & Suyama, A. T. (1975). Inhibition of replication of normal adrenocortical cells in culture by adrenocorticotropin. Proceedings of the National Academy of Sciences of the U.S.A. 71, 50735077. Rhodin, J. A. G. (1971). The ultrastructure of the adrenal cortex of the rat under normal and experimental conditions. Journal of Ultrastructural Research 34, 23-71. Ross, R. (1971). The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers. Journal of Cell Biology 50, 172-186. Shima, S. & Pincus, G. (1969). Effects of adrenocorticotrophic hormone on rat adrenal corticosteroidogenesis in vivo. Endocrinology 84, 1048-1054. Slavinski, E. A. & Auersperg, N. (1976). The role of extracellular matrix in controlling the phenotypic expression of normal adult rat adrenal cortical cells in vitro. Journal of Cell Biology 70, 314a. Slavinski, ., Auersperg, . & Juli, J. W. (1974). Propagation in vitro of functional rat adrenal cortical cells: modifications of the differentiated state by culture conditions. In Vitro 9, 260-269.

Slavinski, . ., Juli, J. W. & Auersperg, N. (1976). Steroidogenic pathways and trophic response to adreno¬ corticotrophin of cultured adrenocortical cells in different states of differentiation. Journal ofEndocrinology 69, 385-394.

Stark, E., Gyevai, ., Bukulya, B., Szabo, D., Szalay,

K. S. Z. & Mihaly, K. (1975). Interrelationship between corticosterone production and fine structure in the fetal adrenal cortex. Journal of General and Comparative Endocrinology 25, 472^t86. Suyama, A. T., Long, J. A. & Ramachandran, J. (1977). Ultrastructural changes induced by ACTH in normal adrenocortical cells in culture. Journal of Cell Biology 72, 757-763. Trelstad, R. L., Hay, E. D. & Revel, J. P. (1967). Cell contact during early morphogenesis in the chick embryo. Developmental Biology 16, 78-106.

DESCRIPTION OF PLATES 1 are electron micrographs.)

(All figures except for Fig.

Plate 1

Fig. 1. Effects of culture conditions on the morphology and growth pattern of adrenocortical cells, (a) Primary culture, medium supplemented with foetal calf serum. Epithelial cell monolayer, characteristic of explanted cortical parenchymal cells. Fibroblastic cells in upper right hand corner, (b) Secondary culture in medium supplemented with foetal calf serum after trypsin dissociation. All cells show fibroblastic morpho¬ logy and intercellular organization, (c) Secondary culture in medium supplemented with horse serum after mechanical dissociation. Epithelial-like morphology and intercellular organization, some formation of multilayers, large refractile lipid inclusions. (Phase-contrast optics, 200.) Fig. 2. Secondary culture of fibroblastic cells, grown without ACTH. Wide intercellular spaces, distended rough endoplasmic reticulum (Er), lipid (L) and glycogen aggregations (G). (x 33 000.) Plate 2

Secondary cultures of fibroblastic cells grown without ACTH. Fig. 3. Extracellular material, (a) Cells are coated with discontinuous basement membrane (arrows). (x 15 000.) (b) Masses of fibrillar extracellular matrix, with apparent structural integrity near cell process (arrow), (x 13 000.) Fig. 4. Extensive cytofilaments appear continuous with extracellular space. Note mitochondria with lamellar cristae. ( 13 000.) Fig. 5. Focal thickenings (arrows) within accumulation of cytofilaments. (x 11 000.) Plate 3

Secondary cultures of fibroblastic cells.

Fig. 6. Gap junctions, (a) Culture without ACTH: 7-9 nm gap and central lamina consisting of electrondense particles spaced at a distance of about 8 nm. ( 220 000.) (b) Treated with ACTH for 1 day: gap junction between two cell processes (arrow). ( 32 000.) Fig. 7. Treatment with ACTH for 3 days. Increased amounts of smooth endoplasmic reticulum and Golgi complexes, reduction in cytofilaments and extracellular matrix. ( 32 000.) Plate 4 Secondary cultures of epithelial cells grown without ACTH. Fig. 8. Cells with large lipid inclusions. Some rough endoplasmic reticulum and Golgi complexes, many mitochondria with lamellar cristae and electron-dense matrix bodies (arrow). Cells are closely adherent. Many pinocytotic vesicles at the cell surfaces. ( 21 000.) Fig. 9. Mitochondria with tubulolamellar cristae. Prominent smooth endoplasmic reticulum arranged, in part, concentrically around the mitochondria. ( 48 000.) Fig. 10. Long, narrow, crystal-shaped space within a lysosome, suggestive of the location of a cholesterol crystal before fixation. Arrows indicate lysosomal membrane. ( 28 000.) Plate 5 Secondary cultures of epithelial cells. Fig. 11. Without ACTH. Numerous electron-dense mitochondrial matrix bodies. ( 40 000.) Fig. 12. Treatment with ACTH for 1 day. Compared with Fig. 8, cells have retracted and there are fewer pinocytotic vesicles and lipid inclusions, (x 16 000.) Fig. 13. Treatment with ACTH for 3 days. Extensive vesicular smooth endoplasmic reticulum and dilated Golgi complexes. ( 24 000). Plate 6 Fig. 14. Secondary culture of epithelial cells after treatment with ACTH for 5 days. The cells have rounded and retracted from one another. Extensive smooth endoplasmic reticulum, few polysomes and membrane associated ribosomes, few small lipid inclusions, (x 16 000.)

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Cultured adrenocortical cells in various states of differentiation: electron microscopic characterization and ultrastructural response to adrenocorticotrophin.

CULTURED ADRENOCORTICAL CELLS IN VARIOUS STATES OF DIFFERENTIATION: ELECTRON MICROSCOPIC CHARACTERIZATION AND ULTRASTRUCTURAL RESPONSE TO ADRENOCORTI...
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