Fine structure of adrenal cortex in rats harbouring a medullary thyroid carcinoma transfected with a corticotrophin-releasing hormone cDNA expression vector
M. Feinmesser, S. L. Asa, K. Kovacs and M. J. Low*
Department of Pathology, St Michael’s Hospital, University of Toronto, Toronto, Ontario M5B 1W8, Canada *Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, Oregon 97201, U.S.A.
RECEIVED 27 January 1992
ABSTRACT
We report the light microscopic, transmission and scanning electron microscopic features of the adrenal cortices in rats bearing a medullary thyroid carcinoma cell line transfected with a corticotrophin-releasing hormone (CRH) cDNA expression vector. The ani- mals had elevated CRH, ACTH and corticosterone blood levels, involuted thymuses and markedly enlarged adrenal glands with prominent lipid-depleted cortices and dilated congested capillaries, similar to those of animals treated with ACTH. Using electron microscopy it was found that the enlarged fasciculata and reticularis zones were composed of large, compact cells with abundant smooth endoplasmic reticulum, prominent Golgi complexes, increased number of large mitochondria with focal loss of cristae and cavit- ation of the internal compartments, numerous lyso- somes and prominent elongated microvilli. In addition, small cytoplasmic fragments were seen within the capillary lumina; these structures resem- bled microvilli that were apparently detached from
adrenocortical cells and entered the blood stream via discontinuous endothelium of dilated capillaries. By scanning electron microscopy it was found that the cells had bulging surfaces with scattered pits and numerous long microvilli pointing in different directions.
This animal model allows analysis of the effects of protracted CRH excess resembling tumoural CRH- dependent Cushing’s syndrome in human patients. Our findings call attention to the role of microvilli in adrenocortical secretion. The increased number and size of microvilli has been thought to lead to an increase in the surface area of adrenocortical cells, thereby facilitating hormone discharge. The detach- ment of microvilli from adrenocortical cells may rep- resent a form of apocrine secretion and may contribute to hypercorticosteronaemia in CRH excess.
Journal of Endocrinology (1992) 135, 271-277
INTRODUCTION
Corticotrophin-releasing hormone (CRH), a 41 amino acid hypothalamic peptide, increases cortico- trophin (ACTH) secretion from the pituitary (Vale, Spiess, Rivier & Rivier, 1981) and possibly also from extrahypophysial sources (Fehm, Klein, Holl & Voigt, 1984; Fehm, Holl, Späth-Schwalbe et al. 1988; Bornstein, Ehrhart, Scherbaum & Pfeiffer, 1990). A variety of tumours has been reported to contain CRH, often in association with ACTH (Carey, Varma, Drake et al. 1984; Zárate, Kovacs, Flores et al. 1986; Asa, Kovacs, Vale et al. 1987; Jessop, Cunnah, Millar et al. 1987). Some of the patients
develop Cushing’s syndrome with documented hyper- cortisolaemia and adrenocortical hyperplasia.
An excess of CRH can be induced experimentally in animals. It has been shown in rats that administra- tion of CRH stimulates the pituitary (Gertz, Contreras, McComb et al. 1987), causes increased blood ACTH and corticosterone levels, and enlarge- ment of the adrenals. The morphological changes in the adrenals due to chronic ACTH stimulation have been studied (Kovacs, Horvath, Singer & Lilienfield, 1977; Nussdorfer, 1986; Andreis, Rebuffat, Belloni et al. 1989; Li, Asa, Kovacs et al. 1990); however, to our knowledge the morphological features of the
Journal of Endocrinology (1992) 135, 271-277 C) 1992 Journal of Endocrinology Ltd Printed in Great Britain 0022-0795/92/0135-0271 $02.00/0
adrenals of animals with prolonged CRH excess have not been reported.
In the present work, a rat medullary thyroid carcin- oma cell line was transfected with the CRH gene and the tumour cells implanted subcutaneously into host animals. The tumours grew rapidly, resulting in pro- found stimulation of the pituitary-adrenocortical axis. In this paper we report the morphological findings in the adrenal cortices of the animals utilizing light microscopy, transmission and scanning electron microscopy.
MATERIALS AND METHODS
Experimental animals
Sibling male WAG/Rij Wistar rats were the offspring of a single inbred mother obtained from Dr B. Roos (University of Miami, Miami, FL, U.S.A.) (Roos, Yoon, Frelinger et al. 1979); they were maintained in American Association for Accreditation of Labora- tory Animal Care (ACLAC)-certified housing under a 12 h darkness: 12 h light cycle with lab chow and water available ad libitum. All animal studies were conducted in accordance with the NIH guidelines for the use and care of laboratory animals.
Tumour cell line
The rat medullary thyroid carcinoma cell line W2 (also termed WE 4/2) (Haun, Beinfeld, Roos & Dixon, 1989) was the gift of Dr B. Roos. A rat CRH expression vector was constructed that contained pro- moter/enhancer sequences from the human cyto- megalovirus immediate early gene, CRH coding sequences from a rat CRH cDNA, and intron splice/ polyadenylation signals from the SV40 small t antigen gene as described previously (Hammer, Mueller, Liu et al. 1992). The resulting plasmid pCRH-BS was cotransfected with pRSV-neo into W2 MTC cells using the calcium phosphate precipitation technique and individual G418 resistant clones were screened for CRH expression by radioimmunoassay (RIA). Positive clones were amplified and passaged; clone W2CRH-7 was used for the experiment described in this report.
Implantation of tumours
At 5 weeks of age, the host male WAG/Rij Wistar rats were anaesthetized with 2% (w/v) tribromo- ethanol in water (0.15 ml/10 g body weight i.p.) and injected subcutaneously over the left shoulder blade with 6 x 106 freshly trypsinized cells of the W2CRH- 7 line resuspended in 0-5 ml 0-9% (w/v) NaCl. Male age-matched controls received non-transfected W2 tumour implants or no implants. Rats were killed by
decapitation 10-15 weeks after implantation. The adrenal glands of twelve animals bearing W2CRH tumours, seven animals with control W2 tumours and twelve control animals not bearing tumours were sub- mitted for morphological examination.
Morphologic methods
For histology the adrenals were fixed in 10% buffered formalin and embedded in paraffin wax. Three sec- tions (5 um thick) of each gland were stained with haematoxylin and eosin. For electron microscopy small pieces of tissue were fixed in 2.5% (v/v) glutaral- dehyde, post-fixed in 1% (w/v) osmium tetroxide, dehydrated in graded ethanols and embedded in Epon-Araldite (Marivac Ltd, Halifax, NS, Canada). Two ultrathin sections of each gland were stained with uranyl acetate and lead citrate and examined with a Philips 410LS transmission electron microscope; 10- 25 photomicrographs of each grid were further exam- ined in detail. For scanning electron microscopy, glut- araldehyde-fixed and osmicated tissue was dried at the critical point of CO2, mounted and sputter-coated with gold and examined with a Philips S515 scanning electron microscope.
RESULTS
The stably transfected W2CRH cell line secreted CRH which was shown to have biological activity in vitro and in vivo and the details are provided elsewhere (Hammer et al. 1992). In brief, the animals bearing W2CRH tumours had significantly elevated pituitary ACTH and B-endorphin content and elevated circu- lating plasma levels of CRH, ACTH and cortico- sterone (Hammer et al. 1992). From 11 to 16 weeks after tumour implantation, corticosterone ranged from 21 to 28 µg/1 in control and W2-treated animals and from 108 to 285 µg/1 in animals bearing W2CRH tumours. There was complete cessation of body growth within 2 weeks of W2CRH tumour implanta- tion; at 1 month after implantation, the control and W2-treated rats appeared to be healthy but the W2CRH-treated rats had actually lost weight. By 2 months, the control rats bearing W2 tumours had also lost weight, possibly because of their primary tumour burden. At autopsy, animals bearing W2CRH tumours had markedly reduced thymic weights; con- trol rats bearing non-transfected W2 tumours had slightly reduced thymus weights.
Adrenal weights
At autopsy, animals bearing W2CRH tumours had enlarged adrenal glands. Control rats bearing non- transfected W2 tumours had only slightly increased
adrenal weights compared with intact controls. Con- trol adrenal weights (left and right combined) ranged between 36 and 40 mg. The adrenals from rats har- bouring W2 tumours ranged between 44 and 54 mg. The adrenals of rats harbouring W2CRH tumours weighed between 65 and 99 mg.
Light microscopy
The adrenals of control rats and rats transplanted with the control W2 tumour had a similar appearance. The zona glomerulosa consisted of approximately seven to eight layers of compact cells, containing round nuclei with finely dispersed chromatin and inconspicuous nucleoli and moderate amounts of pale, foamy cytoplasm. The cells of the inner cortical zones contained round vesicular nuclei with promin- ent nuclear membranes and conspicuous nucleoli. The cytoplasm varied from lightly eosinophilic and granu- lar to chromophobic and foamy (Pl. 1, Fig. 1). The adrenals of animals bearing W2 tumours contained fewer cells with clear, foamy cytoplasm and a pre- dominance of cells with eosinophilic granular cyto- plasm. Capillaries throughout the cortex were focally dilated but there was no major congestion and no haemorrhages were seen.
The adrenals of all 12 animals with W2CRH tumours differed considerably from those of control and W2-bearing animals; the difference was so striking that the glands could be distinguished by blinded observers. The zona glomerulosa appeared thin and attenuated due to the marked hypertrophy and hyperplasia of the inner cortical zones. The zona fasciculata and reticularis were arranged in irregular cords, separated and surrounded by dilated, con- gested capillaries. Few scattered microhaemorrhages were present, mainly in the inner cortex. Nuclei con- tained pale finely dispersed chromatin and large baso- philic nucleoli. The cytoplasm was abundant, eosinophilic and distinctly granular (Pl. 1, Fig. 2). Only occasional cells at the periphery of the gland had pale foamy cytoplasm.
Transmission electron microscopy
In control animals, glomerulosa cells possessing char- acteristic mitochondria with lamellar cristae could be identified. The nuclei of cells in the zona fasciculata and reticularis were slightly irregular with some con- densation of the chromatin along the nuclear mem- brane; occasional nucleoli were noted. The cytoplasm (Pl. 1, Fig. 3) was filled with lipid vacuoles and numer- ous spherical mitochondria which varied in size and shape; their cristae were tubulovesicular with occa- sional paracrystalline structures. There were loosely packed vesicles and tubules of smooth endoplasmic
reticulum; rough endoplasmic reticulum was sparse but free ribosomes were present. The Golgi apparatus and lysosomes were prominent. The cells were closely apposed with well-defined cell membranes. Microvilli in the barely discernable intercellular spaces were few, however, in the perivascular spaces microvilli were clearly evident; they were short, broad and oriented parallel to the capillary basement membrane (Pl. 2, Fig. 5). Capillary endothelial cells with scattered small fenestrations rested on an intact basement membrane. There was no increase in collagen or ground substance.
The ultrastructural features of adrenal cortices from animals bearing W2 tumours were similar to those of control animals with a few minor differences. Lipid vacuoles were less numerous. The intercellular spaces were slightly dilated with a moderate number of finger-like microvilli oriented at right angles to the cell membranes; microvilli were particularly promin- ent in perivascular spaces.
In adrenals of rats bearing W2CRH tumours, glomerulosa cells possessing characteristic mitochon- dria with lamellar cristae were identified. The fascicul- ata cells of all 12 animals appeared larger than in normal glands and possessed round to oval nuclei with markedly irregular nuclear outlines and thin rims of condensed chromatin at the periphery. Most nuclei contained one or more nucleoli. The cytoplasm was abundant and packed with numerous cytoplasmic organelles consisting almost exclusively of mitochon- dria and well developed, tightly packed, vesicular, smooth endoplasmic reticulum (Pl. 1, Fig. 4). The mitochondria were round to oval with marked varia- tion in size and shape and prominent moulding; giant forms were readily seen. Their cristae were vesicular or tubulovesicular. Some mitochondria contained paracrystalline structures and showed focal cavitation of their internal compartments with loss of cristae. The Golgi apparatus was conspicuous. Lipid was depleted but electron-dense lysosomal bodies were more numerous than in control and W2-bearing ani- mals. The cell membranes were well defined. Numer- ous long microvilli protruded into the focally dilated intercellular and perivascular spaces. The microvilli were finger-like, wavy, interdigitating and oriented vertical to the cell surface (Pl. 2, Fig. 6). Capillaries were markedly dilated and there were discontinuities in the endothelium as well as in basement membranes. In other areas, endothelial fenestrae were intact and seemed to be larger than in controls; a few endothelial pockets were seen. Within the capillary spaces there were small membrane-bound fragments of cytoplasm resembling microvilli (Pl. 2, Figs 7 and 8). The frag- ments were round to oval or elongated and were pre- sent both in the centre and at the periphery of the
capillaries. No increase in collagen, extracellular fibrils or ground substance was observed.
Scanning electron microscopic appearance
In control animals and those bearing W2 tumours, the adrenocortical cells were of medium size with bulging cell surfaces. Microvilli were few, scattered and stubby (Pl. 3, Fig. 9). Adrenals from animals bearing W2CRH tumours had large bulging cell sur- faces with numerous long microvilli pointing in different directions (Pl. 3, Fig. 10). Scattered pits were present on the membrane surface.
DISCUSSION
The results of our study prove that protracted CRH stimulation induces adrenocortical hyperplasia simi- lar to that occurring during ACTH stimulation (Kovacs et al. 1977; Andreis et al. 1989; Nussdorfer, 1986; Li et al. 1990). The ultrastructural features of the enlarged adrenocortical cells from animals bearing W2CRH tumours include lipid depletion, an increased number of microvilli and mitochondria, the latter with cavitation of the internal compartment, and hyperplasia of smooth endoplasmic reticulum. These are considered to be the morphological corre- lates of increased hormonal stimulation resulting in enhanced corticosteroid secretion. The enzymes required for corticosteroid synthesis have been loca- ted in mitochondria and smooth endoplasmic reticu- lum (Nussdorfer, 1986; Andreis et al. 1989). Our study provides a valuable model for the analysis of the effects of protracted CRH excess resembling tumoral CRH-dependent Cushing’s syndrome in human patients.
Over the years several theories have been proposed concerning the mechanism of corticosteroid secretion. The holocrine secretion theory has only historical value (Nussdorfer, 1986). The endoplasmocrine secre- tion theory proposed by Rhodin (1971) suggested that steroids were stored in lipid vacuoles and released into the extracellular spaces in the form of a smooth endoplasmic reticulum-lipid droplet complex. The exocytotic secretion hypothesis has gained support in recent years (Nussdorfer, 1986). Morphological analysis of the adrenal glands of several species has demonstrated the presence of dense membrane-bound intracellular structures in the zona fasciculata, often increasing with ACTH administration, and located near intercellular canaliculi, opening into the suben- dothelial space (Gemmell, Laychock & Rubin, 1977; Mazzocchi, Belloni, Rebuffat et al. 1979; Boshier, Holloway & Liggins, 1980). A striking increase in the number of these electron-dense bodies was noted in
the zona glomerulosa of hypertensive rats (Rebuffat, Belloni, Mazzocchi et al. 1979). Nevertheless, exocy- totic release of their contents was never actually observed (Gemmell et al. 1977; Mazzocchi et al. 1979; Rebuffat et al. 1979). Secretion of steroids by simple diffusion is a widely accepted theory (Nussdorfer, 1986; Setoguti, Inoue & Shin, 1987). Scanning elec- tron microscopy demonstrates a large number of elon- gated branching finger-like microvilli, predominantly on the plasma membranes of cortical cells facing capillaries in ACTH-stimulated adrenals, in contrast to short wide microvilli in non-treated animals or humans or in hypophysectomized rats (Setoguti et al. 1987; Li et al. 1990). The larger surface area would facilitate uptake of substances from the extracellular space and discharge by diffusion of steroids from the cells.
The presence of what appear to be small cyto- plasmic fragments within the lumina of capillaries apparently detached from adrenocortical cell bodies requires further analysis and interpretation. The simi- larity of these fragments to the tips of microvilli in the perivascular space suggests the possibility that microvilli break off and enter into the blood stream.
Cell fragments noted within adrenal capillary lum- ina in our studies suggest the possibility of a form of apocrine secretion in the adrenal cortex. Brenner (1966) first observed degenerating cytoplasmic projec- tions extending from the subendothelial space, trav- ersing annular pores of endothelial cells and extending into the vascular lumen in the monkey zona glomeru- losa. Subsequently, Penney, Averill & Olson (1972) and Penney, Olson & Averill (1973) described appar- ently viable cytoplasmic fragments within capillary lumina in continuity with parenchymal cells in the adrenal cortex of rats administered prostaglandin E, or E2; however, those authors reported that the cyto- plasmic fragments were not more prominent in chron- ically stressed or ACTH-stimulated animals than in controls, suggesting that they did not represent apoc- rine secretion by adrenocortical cells (Penney et al. 1972). It is well documented that microvilli are more prominent, numerous and elongated in ACTH-stimu- lated adrenocortical cells (Nussdorfer, 1986) and their prominence around capillaries has been stressed (Brenner, 1966; Pudney, Sweet, Vinson & Whitehouse, 1981; Matsuo & Tsuchiyama, 1987). On transmission and scanning electron microscopy, the adrenals of rats harbouring W2CRH tumours demon- strated a large number of microvilli mainly around blood vessels. The microvilli were thin, branching and predominantly perpendicular to the cell membrane in contrast to the wide, short microvilli lying parallel to the cell membrane in control animals. Cytoskeletal microtubules and actin filaments are thought to play an important role in hormone secretion (Nussdorfer,
1986; Loesser & Malamed, 1987); actin filaments which have been shown to increase in the peripheral cytoplasm and microvilli of stimulated adrenocortical cells (Nussdorfer, 1986; Loesser & Malamed, 1987) may provide the erectile apparatus to alter the shape and position of microvilli. The microvilli with narrow bases could protrude into capillaries through discon- tinuities in the endothelium and basement membrane whereas the short, stubby microvilli of controls would not protrude as easily into blood vessels and the broad base would not be broken off with ease. The cause and mechanism of detachment of microvilli remains uncertain.
The capillaries must be involved in this process, since intact basement membrane and endothelium would act as a barrier to the entry of cellular frag- ments into the vascular lumen. It is known that ACTH perfusion of rat adrenal glands causes increased blood flow and marked dilatation of capil- laries and intercellular spaces, with an extensive sys- tem of filopodia closely apposed to blood vessel walls (Pudney et al. 1981). Within 20 min of ACTH treat- ment, there is an increase in size of endothelial fenes- trae from 30-45 nm to 50-80 nm; other endothelial openings, including double diaphragmed channels, transendothelial openings and unknown types of openings, have also been identified after ACTH administration (Apkarian & Curtis, 1986; Apkarian & L’Hernault, 1990). In our study, there were break- age of the capillary basement membranes and discon- tinuities of endothelium. It is known that after chronic exposure to ACTH there is capillary congestion with extravasation of erythrocytes and microhaemorrhages are seen on light microscopical examination; endo- thelial discontinuities implicated in those changes may also be the route of release of microvillus fragments into the capillary lumen.
Coated pits have been considered to be the morpho- logic correlate of endocytosis (Nussdorfer, 1986); however, their prominence adjacent to groups of microvilli raises the possibility that they represent the site of detached microvilli. Matsuo & Tsuchiyama (1987) described a primary glucocorticoid-producing adrenocortical tumour without pits and concluded that they did not participate in steroid release; how- ever, in that case of primary adrenal hyperfunction, both ACTH and CRH were probably markedly sup- pressed. Release of microvilli may be mediated by ACTH and/or CRH.
One must consider that the cytoplasmic fragments may be artifactual, or due to oblique cutting, but our specimens were well preserved and the presence of cytoplasmic fragments in capillaries was a consistent finding in several animals; in addition, cytoplasmic fragments were not found in the adrenals of control rats. It may be that they are the results of hypoxia or
endothelial damage with the development of discon- tinuities in the vascular wall, but there was no evi- dence of ischaemic damage in these tissues. It is also possible, although unlikely, that the cytoplasmic frag- ments may represent endothelial processes, but no continuity was found between the endothelial cell cytoplasm and the fragments. In studies of normal and ACTH-stimulated rat adrenal cortices, Apkarian & Curtis (1986) and Apkarian & L’Hernault (1990) published transmission and scanning electron micro- graphs of capillary lumina containing spherical struc- tures which may be microvillus fragments and ‘ghost’ profiles abutting against the subluminal aspect of the endothelial cell cytoplasm that resemble our findings. They stressed their non-artifactual presence and thought they were short-lived structures which were involved in the uptake of precursors and release of product from steroidogenic to vascular compart- ments. The structures in the photomicrographs of one of those studies (Apkarian & L’Hernault, 1990) differ from ours in that they appear ghost-like, whereas in our material the fragments contain cytosol similar to that of the adjacent microvilli.
The main argument against our hypothesis is that we have not demonstrated microvilli in the process of extension into the capillary lumen. Further studies using serial sections and tracer particles may resolve this question.
Our findings support the concept of apocrine secre- tion, but it is clear that hormone secretion may be achieved by several means (Bassett & Pollard, 1980; Nussdorfer, 1986). Under normal circumstances, corticosterone may be secreted by simple diffusion, but 18-deoxycorticosterone, the other major product of the zona fasciculata, is not (Nussdorfer, 1986); at high levels of stimulation both steroids are probably secreted to some extent by diffusion (Pudney et al. 1981). Stimulating trophic factors may modify secre- tion in a specific manner. Cytoplasmic projections into rat adrenocortical capillaries appeared following prostaglandin administration, a feature not typical of ACTH stimulation (Penney et al. 1972, 1973). It may be that direct CRH stimulation of the adrenals modi- fies secretion in a unique manner which involves a form of apocrine release of microvilli. The secretion hypothesis we present may eventually be added to the long list of hypotheses regarding glucocorticoid secretion. Further work should clarify this intriguing issue.
ACKNOWLEDGEMENTS
This work was supported by grants MT 6349 and MA-10215 of the Medical Research Council of Canada and by grant DK 40457 of the NIH. The
authors thank Dr E. Horvath for advice with the ultrastructural studies, Mrs D. Lietz, Mr R. Logan and Mrs N. Nelson for technical assistance and Ms C. Drvodelic for secretarial help.
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Journal of Endocrinology (1992) 135, 271-277
DESCRIPTION OF PLATES
Plate 1
FIGURE 1. The adrenal cortex of a control rat is composed of clear and compact cells (haematoxylin and eosin stain; ×300). ☒
FIGURE 2. In contrast, the cortex of a rat harbouring a W2CRH tumour is composed of larger cells with abund- ant, pale eosinophilic, granular cytoplasm (haematoxylin and eosin stain; ×300).
FIGURE 3. By electron microscopy, cells of the zonae fas- ciculata and reticularis of the adrenal cortex in a control rat contain prominent lipid droplets (arrow) and numer- ous mitochondria (arrowhead) (bar = 5 um).
FIGURE 4. The corresponding cells in a rat harbouring a W2CRH tumor are depleted of lipid. They have abundant smooth endoplasmic reticulum and numerous mitochond- ria with vesicular cristae and paracrystalline structures (arrows); giant mitochondria are prominent (arrowhead) (bar=5 um).
Plate 2
FIGURE 5. In the adrenal cortex of a control rat, the capil- laries are small, have intact basement membrane and are surrounded by adrenocortical cells with short, broad microvilli lying parallel to the basement membrane (arrows) (bar= 1 um).
FIGURE 6. In the adrenal cortex of a rat with a corticotro- phin-releasing hormone-producing tumour, the dilated capillary is lined by endothelium with prominent fenestrae (arrowheads) and a disrupted basement membrane; the underlying adrenocortical cells have numerous long, finger-like microvilli extending perpendicular to the luminal surface (arrows) (bar= 1 um).
FIGURES 7 and 8. Within the capillary lumina of adrenal cortices of rats harbouring W2CRH tumours, there are small, membrane-bound fragments of cytoplasm (arrows) which are elongated like microvilli (Fig. 7) or round, resembling the cut tips of microvilli (Fig. 8) (bars =0.5 um).
Plate 3
FIGURES 9 and 10. By scanning electron microscopy, the adrenal cortical cells of control rats (Fig. 9) have bulging surfaces with few, stubby microvilli (arrows). In contrast, the corresponding cells in animals bearing W2CRH tumours (Fig. 10) have numerous long microvilli (arrows) and scattered pits (arrowheads) on the cell surfaces (bars =5 um).
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PLATE 3
CRH-stimulated adrenal cortex . M. FEINMESSER and others
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