of Androgen Treatment - Europe PMC

Recovery of Adrenal Ultrastructure After Cessation of Androgen Treatment Peter A. Nickerson, PhD and Agostino Molteni, MD, PhD

Female, uninephrectomized rats injected with methylandrostenediol became hypertensive by 4 weeks of treatment. After cessation of treatment, blood pressure continued to rise during the first week but gradually decreased below the borderline of hypertension by 5 weeks. After methylandrostenediol treatment for 4 weeks and after 1 week of recovery, adrenocortical fine structe exibited a number of abnormalities including reduced numbers of mitochondrial cristae, hypertrophic smooth endoplamic reticulum, microfilaments and large lipid vacuoles within zona reticularis cells. During an intermediate period of recovery (2 weeks), mitochondrial cristae increased in number, although animals remained hypertensive. After 2 weeks, all of the ultrastrutural abnormalities seen at early and intermediate periods of recovery were fully reversed, concomitant with a return of systolic blood pressure to below hypertensive levels (Amer J Path 64:31-44, 1970).

TREATrMENT OF FEMALE, UNINEPHRECTOMIZED SALT-DRINKING

RATS with the synthetic androgen methylandrostenediol (MAD) produces a severe form of cardiovascular disease characterized by marked elevation of systolic blood pressure.' The adrenal gland is directly involved in the pathogenesis of the hypertensive disease since adrenalectomized animals do not develop elevation in blood pressure.2 Adrenocortical dysfunction in androgen-treated animals resulting in amounts of desoxycorticosterone (DOC) above control levels in adrenal venous blood3 and in incubations of adrenocortical tissue ' suggests that this steroid is involved at least in part in the evolution of the hypertension. Ultrastructural abnormalities in adrenocortical tissue accompany adrenal dysfunction. Among these alterations are reduced numbers of mitochondrial cristae,46 occurrence of vacuolar inclusions,7 droplets 7 and cytoplasmic microfilaments,S and hypertrophv of smooth endoplasmic reticulum.48 Preliminary studies from our laboratory have suggested that blood pressure changes in animals treated intraperitoneally with androgen for as short as 4 weeks are reversible when treatment is stopped and the animals allowed to recover from the effects of the androgen.9 No conFrom the Department of Pathology, State Universitv of New York at Buffalo. Supported by grant HE 06975 from the National Heart Institute and training grant 01500 from the National Institute of General Medical Sciences. Accepted for publication March 12, 1971. Address for reprint requests: Dr. Peter A. Nickerson, Department of Pathology, 204 Capen Hall, State University of New York at Buffalo, Buffalo, New York 14214. 31

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American Journal of Pathology

comitant studies of the ultrastructure of the adrenal gland during the recovery period have appeared. In view of the essentiality of the adrenal gland in the pathogenesis of the hypertension,2 it was the purpose of the present investigation to study the sequential changes in the adrenal gland and blood pressure to determine whether these parameters are reversible during recovery from androgen treatment. Materials and Methods Forty-two female Charles River CRR rats 43 days of age were uninephrectomized and allowed to rest for 2 days before injections were started. All animals were caged individually and housed in a room maintained at 22 C. Animals were given Purina Lab Chow ad libitum and 1% sodium chloride as drinking solution throughout the entire 9 weeks of the experiment. Twenty-four animals were injected subcutaneously 5 days/week for 4 weeks with 10 mg methylandrostenediol (Nutritional Biochemicals, Cleveland, Ohio) made up as a microcrystalline suspension in distilled water.1 The remainder of the animals served as controls and received subcutaneous injections of distilled water. Skelton1 has shown that severe hypertensive cardiovascular disease is produced by this regimen. Injections were stopped after 4 weeks, although animals continued to receive 1% saline as drinking fluid. Four androgen-treated animals and 3 controls were sacrificed by decapitation for electron microscopic examination at 4 weeks of injections and at 1, 2, 3, 4 and 5 weeks after injections were stopped. Blood pressures were measured by plethysmography of the tail and recorded on an E&M physiograph 4 (E&M Instruments, Houston, Texas). Measurements of blood pressure were made in lightly anesthetized animals before injections were begun, after 4 weeks of treatment and at 2 and 5 weeks after recovery from injections. After decapitation of the animals, adrenals were removed quickly and trimmed of adherent fat. At each time period, 4 experimental and 3 control adrenals were cut into slices approximately 1 mm in thickness and fixed in 3% purified glutaraldehyde (Ladd Research Industries, Burlington, Vermont) buffered to pH 7.3 with 0.1 M phosphate. After fixation, the slices were washed in several changes of ice-cold buffer. While still in buffer, the three cortical zones were separated with a microscalpel under a dissecting microscope. The zona glomerulosa was isolated by taking thin tangential sections adjacent to the capsule. The zona fasciculata was distinguished from the zona reticularis by the whitish color of the former cells in contrast to the reddish color of the latter ones. Tissues were postfixed in 1% osmium tetroxide buffered to pH 7.2 with 0.1 M phosphate. After dehydration in a graded series of ethanol, the adrenals were embedded in Epon 812 and Araldite.10 Before thin sections were cut from a block, 1-ft-thick sections first were cut and stained with toluidine blue to verify the location of the tissue blocks. Thin sections were cut with glass knives on a Porter-Blum ultramicrotome. Sections were stained with methanolic uranyl acetate 11 and lead citrate 12 before examination with a Siemens 101 electron micro-

scope.

Results

MAD injections induced a marked rise in blood pressure along with gross lesions in the heart, kidney and mesenteric blood vessels, which were identical to those reported previously.1 Blood pressure values in

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MAD-treated and control animals were not significantly different before injection with values of 112.3±+ 1.9 and 106.9±+-3.8 nunng, respectively. The blood pressure of androgen-treated animals was 180.1+3.1 mmHg after 4 weeks and 122.6±+3.8 mmHg for controls. After 2 weeks of recovery, blood pressure for androgen-treated animals was 160.5±+ 6.0 mmHg whereas that for controls was 120.1±3.6 mmHg; after 5 weeks of recovery, the values were 140.3±6.0 and 124.0±6.7 mmHg, respectively. The periods of recovery after cessation of MAD injections can most conveniently be divided into three periods, consisting of early, intermediate and late times, based upon differences in adrenal ultrastructure. All changes to be described occurred in fasciculata-reticularis cells whereas gomerulosa cells were identical morphologically to those in controls. Mrenocotcal Fine Stucture in Control

No observable morphologic changes were observed in adrenocortical cells of uninephrectomized, salt-drinking controls when compared to untreated, normal animals. Since the adrenocortical fine structure in controls was similar to that described previously,"3 14 the ultrastructure of control adrenocortical cells will not be described in detail. It should be emphasized however that mitochondria in fasciculata-reticularis cells have tubulovesicular cristae which virtually fill the entire mitochondrial matrix. The connection of cristae with the inner mitochondrial membrane is not frequently demonstrable. Furthermore, fasciculata-reticularis adrenocortical cells possess predominandy smooth endoplasmic reticulum. No microfilaments and only an occasional microtubule are observable within cortical cells. Early Perod of Recey

The time between 4 weeks of injections and 1 week after cessation of MAD injections comprises the initial period of recovery. Alterations in adrenocortical fine structure after 4 weeks of injections were similar to those reported by Levine and Skelton 7 after 8 weeks of MAD treatment. A reduced number of mitochondrial cristae was particularly evident in androgen-treated animals (Fig 1). Many mitochondria possessed cristae resembling simple tubules that were connected with the inner mitochondrial membrane, unlike cristae of control mitochondria. One week after cessation of injections, alterations in mitochondrial cristae were similar if not more severe than after 4 weeks of continuous treatment. A large proportion of adrenocortical cells contained mito-

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chondria whose cristae were reduced in number and located peripherally (Fig 2). There was a marked hypertrophy of smooth endoplasmic reticulum at both time periods (Fig 1 and 2); focal accumulations of smooth reticulum were often arranged in a whorl-like configuration. Cisternae of rough endoplasmic reticulum were few in number in cells with hypertrophic smooth endoplasmic reticulum. Polyribosomes, however, were dispersed randomly throughout the cytoplasm. Microfilaments were observed at 4 weeks of injections and even more frequently after 1 week of recovery (Fig 2). Bundles of the filaments composed of between 3 and 6 filaments frequently traversed the cytoplasm at random. Filaments appeared to accumulate in a paranuclear region at 1 week of recovery. Lysosomes with granular, lucid or electron-dense inclusions were especially numerous in fasciculata-reticularis cells at both time periods (Fig 1 and 2). Lipid droplets varied in size within cells of inner cortical zones. Droplets in fasciculata cells were generally small and few in number; the matrix of the droplets consisted of an amorphous, evenly distributed flocculent material (Fig 2). On the other hand, lipid droplets in reticularis cells were frequently so large that only one was found per cell, suggesting that several droplets had coalesced into one large droplet. The droplets showed a flocculent matrix similar to that in fasciculata cells. Neither cytoplasmic vacuoles nor hyaline droplets, reported after 8 weeks of androgen injections by Levine and Skelton,7 were observed in the inner cortical zones. Intermediate Period of Recovery

Two weeks after cessation of MAD injections, adrenocortical fine structure began to return to that of controls and thus it seemed appropriate to characterize this time as an intermediate period of recovery. Mitochondria in many fasciculata-reticularis cells were larger in size and contained an increased number of cristae as compared with mitochondria during the initial stage of recovery (Fig 3). However, the cristae in mitochondria in adjacent cells still were reduced in number as in the initial recovery period. Smooth endoplasmic reticulum was hypertrophic (Fig 3), although no whorls of smooth membranes were observed in adrenocortical cells. Similarly, microfilaments were not as numerous as at 1 week of recovery. Several randomly oriented microtubules were first observed at this time period. The small lipid droplets usually seen in controls were few in number in zona fasciculata cells. Similarly, the number of enlarged droplets observed during the initial period of recovery was reduced in reticularis cells. Lysosomes were prominent in most adrenocortical cells.

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The Golgi apparatus was prominent, consisting of several cisternae and associated vesicles. Late Pd of

y

A late stage of recovery was observed between 3 and 5 weeks after cessation of androgen injections (Fig 4-6). The proportion of cells with mitochondrial cristae similar to those of controls increased between 3 and 5 weeks, such that by 5 weeks, mitochondria could not be readily distinguished from those of controls (Fig 6). The smooth endoplasmic reticulum remained hypertrophic at 3 weeks of recovery (Fig 4), but decreased at 4 weeks (Fig 5) and was similar to that of controls by 5 weeks (Fig 6). Lysosomes were numerous at 3 and 4 weeks (Fig 5) but decreased in number by 5 weeks. The Golgi apparatus was particularly prominent at 3 and 4 weeks (Fig 4 and 5). Microfilaments were observed in one or two cells after 3 weeks, although no ifiaments appeared in adrenocortical cells at 4 and 5 weeks. The number of lipid droplets per cell increased and the size of these structures decreased during the recovery period. However, even at 5 weeks, the number of lipid droplets was smaller than that in adrenocortical cells of controls. Discussion

The effect of MAD on adrenal ultrastructure was entirely reversible and blood pressure returned to below hypertensive levels after injections of the androgen were stopped. Recovery has been divided into early, intermediate and late periods, according to differences and extent to which the adrenal cortex resembled that of controls. At least two mechanisms seem probable to explain the return of adrenocortical cells in MAD-treated animals toward normal. The abnormal cortical cells could be replaced by new cells through cellular migration, presumably originating in the zona glomerulosa or subglomerulosal region.15'16 Another possibility is that recovery occurs for the most part by repair of existing adrenocortical fasciculata-reticularis cells. All of the observations in the present study support the latter mechanism. In the early stage, mitochondrial cristae were reduced in number. These ultrastructural abnormalities in mitochondria were reflected in alterations in steroid synthesis reported previously from our laboratory 4,5 in which conversion of progesterone to corticosterone by adrenal homogenates is impaired by 4 weeks of MAD treatment. Normalization of steroid biosynthesis, blood pressure and sodium intake levels have also been observed after 4 weeks of MAD injections and recovery for up to 6 weeks.' It should be noted that the conversion of progesterone to

36



corticosterone by adrenal homogenates is a two-step reaction. Progesterone is hydroxylated at position 21 to deoxycorticosterone (DOC) by enzymes in the smooth endoplasmic reticulum.17 This reaction appears to be normal in MAD-treated animals and this is correlated in the present study with hypertrophic smooth endoplasmic reticulum in androgeninjected animals. In a second step, DOC is changed to corticosterone by mitochondrial enzymes.'8 The reaction involves hydroxylation at position 11 of DOC. The 11-3-hydroxylation system occurs on the inner cristae of the mitochondria 19 and this fact accords well with reduced numbers of cristae seen ultrastructurally. Previous studies from our laboratory have demonstrated that it is this mitochondrial step that is abnormal in MAD-treated animals 5 and that mitochondrial P-450 involved in the 11-f-hydroxylation reaction is reduced in concentration.20 MAD binds competitively to the P-450, thereby blocking effective ultilization of the DOC substrate and resulting in inefficient conversion of DOC to corticosterone. Start of the repair process is observed almost simultaneously at all levels of the fasciculata-reticularis after the 2 weeks of recovery. The mitochondrial cristae became more prominent. If cellular migration were the principal mechanism in recovery, one would expect a gradual replacement of cells beginning at the periphery rather than a widespread recovery of cells. The question of cell origin in the adrenal cortex has long been controversial,15'21 although the migration of cells from the periphery of the adrenal has gained increasingly wide support since the work of Kahri 22 and of Brenner.16 Brenner 16 has observed that tritiated thymidine is incorporated into peripherally located cells in the mouse adrenal cortex. After 6 weeks, the labeled cells in the mouse adrenal had apparently migrated into the inner cortex. These observations demonstrate that migration takes a considerably longer time than 2 weeks and thus makes it seem doubtful that migration plays a significant part in the recovery of the adrenal. The cellular mechanism through which repair occurs is not well understood, although changes in several cellular organelles seem related to the reparative process. The increased number of lysosomes observed during the intermediate and late stages of recovery may well be involved in the remodeling of adrenocortical cells. The hypertrophic smooth endoplasmic reticulum became less prominent during recovery and may have been degraded by lysosomal enzymes through autophagocytosis. Alternatively, the smooth reticulum could decrease by transformation into rough reticulum as shown by Dallner, Siekevitz and Palade 23 in the liver. However, this mechanism seems unlikely to account for reduction in the smooth reticulum since no significant hypertrophy

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of rough endoplasmic reticulum occurred during the 5 weeks of recovery. Almost every adrenocortical cell contained microfflaments by 4 weeks of androgen treatment although virtually all of the filaments disappeared during recovery. The disappearance of microfilaments may also be attributable to lysosomal degradation of the filaments themselves or to dissociation of ifiaments into subunits before degradation. Alternatively, Nickerson, Skelton and Molteni 8 have suggested that accumulation of microfilaments is attributable to an inhibition of mitosis because of a failure of microfflaments to form the microtubules comprising the mitotic apparatus. It might be expected from this observation that these cells would undergo mitosis upon removal of androgen. It seems unlikely however that all or even a large portion of cells underwent mitosis since adrenal size did not increase noticeably as would be expected if considerable cellular division had occurred. Concomitant with recovery of the adrenal, changes in blood pressure were largely reversed by 5 weeks, with the pressure falling to 140.3 ± 6.0 mmHg. The borderline of hypertension has usually been considered to be 150 mmHg. Gross lesions in the kidney, heart and mesenteric blood vessels after 4 weeks of MAD injections were identical to those described previously." These lesions could not be identified macroscopically by the fifth week of recovery despite continued consumption of 1% saline as drinking solution. The process of repair of the lesions is not known and has not been investigated in the present study. It has been suggested that the development of MAD hypertension as well as other forms of hypertension studied in our laboratory 6,13,24 iS caused at least in part by DOC, a known hypertensinogenic steroid.-5 Elevations of DOC are observed during incubations of adrenocortical homogenates 4 and in the adrenal venous blood 3 during treatment with MAD. Other investigators have also observed that removal of the hypertensinogenic agent reverses the hypertensive cardiovascular disease provided that the disease has not been of too long duration.2627 Recovery of steroid biosynthesis and adrenal ultrastructure was observed in another study from our laboratory in which animals were treated for 12 weeks and allowed to recover for 6 weeks.28 It should be noted that metacorticoid hypertension is produced under these conditions. References 1. Skelton FR: The production of hypertension, nephrosclerosis and cardiac lesions by methylandrostenediol treatment in the rat. Endocrinology 53:492505, 1953

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2. Molteni A, Skelton FR, Brownie AC: Effect of adrenalectomy on the development of androgen-induced hypertension. Lab Invest 23:429-435, 1970 3. Hyde PM, Daigneault EA: Adrenal plasma levels of corticosterone and deoxycorticosterone in methylandrostenediol-salt induced hypertension. Steroids 11:721-731, 1968 4. Brownie AC, Skelton FR: Adrenocortical function and structure in adrenalregeneration and methylandrostenediol hypertension, Functions of the Adrenal Cortex. Vol 2. Edited by KW McKerns. New York, Appleton-Century-Crofts, 1968, pp 691-718 5. Brownie AC, Skelton FR, Gallant S, Nicholls P, Elliott WB: Adrenal cytochrome levels, corticosteroidogenesis and respiratory activity in the rat following methylandrostenediol treatment. Life Sci 7:765-771, 1968 6. Skelton FR, Brownie AC: Studies on the pathogenesis of adrenal-regeneration and methylandrostenediol hypertension, Endocrine Aspects of Disease Processes. Edited by G Jasmin. St Louis, Warren H Green, Inc, 1968, pp 271301 7. Levine AJ, Skelton FR: A light and electron microscopic study of hyaline droplet and vacuole formation in the adrenal glands of rats treated with methylandrostenediol. Amer J Path 51:831-854, 1967 8. Nickerson PA, Skelton FR, Molteni A: Observation of filaments in the adrenal of androgen-treated rats. J Cell Biol 47:277-280, 1970 9. Molteni A, Brownie AC, Skelton FR: Reversibility of methylandrostenediol hypertension after cessation of androgen treatment. Fed Proc 28:367, 1968,

abstr 10. Mollenhauer HH: Plastic embedding mixtures for use in electron microscopy. Stain Techn 39:111-114, 1964 11. Stempak JC, Ward RT: An improved staining method for electron microscopy. J Cell Biol 22:697-701, 1964 12. Reynolds ES: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208-212, 1963 13. Skelton FR, Brownie AC, Nickerson PA, Molteni A, Gallant S, Colby HD: Adrenal cortical dysfunction as a basis for experimental hypertension. Circ Res 24: Suppl 1:35-57, 1969 14. Nickerson PA, Brownie AC, Skelton FR: An electron microscopic study of the regenerating adrenal gland during the development of adrenal regeneration hypertension. Amer J Path 57:335-364, 1969 15. Skelton FR: Adrenal regeneration and adrenal regeneration hypertension. Physiol Rev 39:162-182, 1959 16. Brenner RM: Radioautographic studies with tritiated thymidine of cell migration in the mouse adrenal after a carbon tetrachloride stress. Amer J Anat 112:81-95, 1963 17. Ryan KJ, Engel LL: Hydroxylation of steroids at carbon 21. j Biol Chem 225:103-114, 1957 18. Brownie AC, Grant JK: The in vitro enzymic hydroxylation of steroid hormones. 1. Factors influencing the enzymic 11 P hydroxylation of 1 -deoxycorticosterone. Biochem J 57:255-263, 1954 19. Dodge AH, Christensen AK, Clayton RB: Localization of a steroid 11 hydroxylase in the inner membrane subfraction of rat adrenal mitochondria. Endocrinology 87:254-261, 1970 20. Brownie AC, Colby HD, Gallant S, Skelton FR: Some studies on the effect

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22. 23. 24.

25. 26.

27. 28.


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of androgens on adrenal cortical function of rats. Endocrinology 86:10851092, 1907 Hoerr N: The cells of the suprarenal cortex in the guinea pig: their reaction to injury and their replacement. Amer J Anat 48:139-197, 1931 Kahri A: Histochemical and electron microscopic studies on cells of the rat adrenal cortex in tissue culture. Acta Endocr (Kobenhavn) 52: suppl 108: 51-96, 1966 Dailner G, Siekevitz P, Palade GE: Biogenesis of endoplasmic reticulum membranes. I. Structural and chemical differentiation in developing rat hepatocyte. J Cell Biol 30:73-96, 1966 Molteni A, Brownie AC, Skelton FR: Production of hypertensive vascular disease in the rat by methyltestosterone. Lab Invest 21:129-137, 1969 Selye H, Hall CE, Rowley EM: Malignant hypertension produced by treatment with deoxycorticosterone acetate and sodium chloride. Canad Med Ass J 49:88-92, 1943 Davis WD Jr, Segaloff A, Jacobs W: The effect of desoxycorticosterone acetate and propylene glvcol in experimental renal hypertension. J Lab Chin Med 33:1483, 1948 Friedman SM, Friedman CL, Nakashima M: Sustained hypertension folowing the administration of desoxycorticosterone acetate. J Exp Med 93:361372, 1951 Molteni A, Nickerson PA, Brownie AC: Unpublished observation, 1970

The authors wish to thank Miss Chistine Szymansk, Mrs. Neonile Fylypiw, Mr. Luther Joseph, Mr. Robert Linsnair and Mrs. Elisabeth Lawson for expert technical assistance.

[IlUutratior foll'

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Fig 1-Zona fasciculata cell after 4 weeks of MAD injections. Mitochondria (M) contain reduced numbers of cristae. Smooth endoplasmic reticulum (SER) is hypertrophic. Lysosomes (LY) are elongate and have a homogeneously electron-opaque matrix. Occasionally, vacuoles (V) are associated with lysosomes. Microfilaments (arrow) are observed throughout the cytoplasm. Microvilli (MV) are seen at the cell surface (x 20,000).

Fig 2-Zona fasciculata cell after 4 weeks of MAD injections and 1 week of recovery. Mitochondria (M) contain few cristae. Numerous lysosomes (LY) contain vacuoles (V) and other electron-dense material. Cross sections (MF) and longitudinal bundles of microfilaments (arrow) traverse the cytoplasm. A lipid droplet (L) contains a flocculent matrix. G indicates Golgi apparatus (x 28,800).

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Fig 3-Zona fasciculata cell at 2 weeks of recovery. Mitochondria contain an increased number of cristae, although many cristae are located peripherally (arrow). Smooth endoplasmic reticulum (SER) is hypertrophic. One or two microfilaments (MF) are observed. Fig 4-Zona fasciculata cell at 3 weeks of LY indicates lysosome (x 27,200). recovery. Mitochondria have numerous tubulovesicular cristae (M). Smooth endoplasmic reticulum (SER) is hypertrophic. The Golgi apparatus (G) is prominent and contains several cisternae and associated vesicles (x 23,200).

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Fig 5-Zona fasciculata cell at 4 weeks of recovery. Mitochondrial cristae (M) are predominantly vesicular. Lyosomes (LY) are particularly prominent and some contain vacuolar structures (V). A focal area of smooth endoplasmic reticulum (SER) is observed. Cisternae of the Golgi apparatus (G) are somewhat dilated (x 28,800). Fig 6-Zona fasciculata cell after 5 weeks of recovery. Mitochondria (M) are almost entirely filled with vesicular cristae. Endoplasmic reticulum is virtually all smooth surfaced (SER), although not hypertrophic. Other structures include a lysosome (LY) and cell membranes (CMJ) of two adjacent cells (x 28,800).

NICKERSON AND MOLTENI ADRENAL RECOVERY AFTER ANDROGEN

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