Zona Reticularis in Aging Spontaneously Hypertensive Rats A Quantitative Ultrastructural Study of 70- and 95-Week-Old Animals Peter A. Nickerson, PhD, Leonard G. Feld, MD, PhD, and Judith B. VanLiew, PhD
The zona reticularis of 70- and 95-week-old female Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats was studied by quantitative stereologic techniques. The zona reticularis in the adrenal gland of 70-week-old SH rats was virtually identical to that of 70-week-old W7KY rats. In both SH and WKY rats at 95 weeks of age, however, there was hypertrophy of smooth endoplasmic reticulum reflected quantitatively in increased volume and surface area, as compared with that of WKY rats or SH rats at 70 weeks of age. Ninety-five-week-old WVKY rats had a significantly greater mitochondrial volume/cell and surface area of mitochondrial membranes than SH rats. Inclusion of lipid droplets within mitochondria was seen in zona reticularis cells from SH and WKY rats at 70 weeks of age; mitochondria-lipid-droplet association was more frequent at 95 weeks of age. The volume of lipofuscin per cell was significantly greater in WVKY than in SH rats at 95 weeks of age. Thus, by quantitative techniques, one can see that the zona reticularis of 95-week-old SH rats differs from that of WKY rats, principally in the presence of smaller cells with a smaller surface area of mitochondrial cristae and a reduced volume of mitochondria, lipofuscin, and lipid droplets. (Am J Pathol 97:433-448, 1979)
OKAMOTO AND AOKI 1 selectively bred Wistar-Kyoto (WKY) rats to form a strain of rats that spontaneously develop hypertension (SH). SH rats develop hypertension,l cardiomegaly, nephrosclerosis,2 vascular disease, and cerebral hemorrhage 3 without any experimental treatment. Abnormalities of the pituitary-adrenal axis,4'5 steroid synthesis, 8 and the thyroid gland9 have been implicated in the pathogenesis of the hypertension, although the essentiality of the endocrine glands in development of the cardiovascular disease has not been proven conclusively.'0 There have been several studies of adrenocortical ultrastructure in SH rats."1-5 Nickerson 13 has shown by quantitative morphologic techniques that the zona glomerulosa and zona fasciculata of 21-week-old male SH rats differ from those of WKY animals. Quantitative morphologic techniques also emphasized the essentiality of using the WKY rat as a control From the Department of Pathology, State University of New York at Buffalo, and the Department of Physiology, Veterans Administration Medical Center, Buffalo, New York. Supported by research grants from the National Heart, Lung and Blood Institute (HL-06975) and the American Heart Association and Veterans Administration Research Funds. Accepted for publication June 29, 1979. Address reprint requests to Peter A. Nickerson, PhD, Department of Pathology, State University of New York at Buffalo, 204 Farber Hall, Buffalo, NY 14214. 0002-9440/79/11 08-0433$01 .00 433 © American Association of Pathologists
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because the adrenal cortex of WKY differs considerably from that of another strain of Wistar rat available in the United States.'3 Most recently, Bartsch et al 14 reported increases in surface area and volume of smooth endoplasmic reticulum in the zona fasciculata from 7-month-old SH rats by stereologic techniques; however, they did not employ WKY rats as controls. Tsuchiyama et al 12 have also examined the adrenal gland of the SH rat and observed an increased number of lysosomes and lipofuchsin pigment in the inner zona fasciculata and zona reticularis in 12-16month-old male rats. The adrenal glands of aging rats have not been studied extensively by electron microscopy. Recently, Nickerson 16 examined the adrenal cortex of retired breeder gerbils and hamsters and reported alterations in the zona reticularis. We therefore wanted to investigate further the zona reticularis in aging WKY and SH rats by quantitative stereologic techniques to assess morphologic alterations by an objective method. Materials and Methods Okamoto SH and WKY female rats were obtained from Laboratory Supply Company, Indianapolis, and raised in animal holding facilities at the Veterans Administration Medical Center. SH and WKY rats were F-generation 42 and 19, respectively. Animals were caged individually and given lab chow and tap water ad libitum. Access to the facility was limited in order to minimize the possibility of introducing infection to the animals.'7 Three rats each of the WKY and SH strains, 70 and 95 weeks of age, were anesthetized with Inactin (Byk Gulden Konstanz). The adrenal gland was removed quickly and trimmed of adherent fat; 1-mm slices were cut and fixed in 3% purified glutaraldehyde (Ladd Research Industries, Burlington, Vt) buffered to pH 7.3 with 0.1 M phosphate. Tissues were washed in 0.1 M phosphate buffer (pH 7.4). While in buffer, radial sections were cut with a microscalpel while at 3x magnification in a dissecting microscope. The sections were cut so as to include a small piece of adrenal medulla, which facilitated the selection and proper orientation of the tissue. Several blocks with hemorrhagic areas and poorly fixed cells were identified by light microscopy and not included in the analysis. Tissues were processed for electron microscopy as described previously.'8 Half-micron-thick sections were cut from tissue blocks, stained with toluidine blue, and examined by light microscopy to confirm the zonal position of the tissue. Thin sections were cut with glass knives on a Porter-Blum MT-1 ultramicrotome, stained with uranyl acetate '" and lead citrate,20 and examined with a Siemens 101 electron microscope. For quantitative techniques, 4 photographs were taken at low (2400X) and high magnification (8000X) from each of 5 tissue blocks for each animal and magnified 3.5X photographically. Magnification was calibrated with a replica grating (Ernest Fullam, Schenectady, NY). Volume and surface area measurements were made on photographic prints according to the method of Weibel and Bolender 2' and had been used previously by our
laboratory.'3 The average volume per adrenocortical cell was determined by the method of Nussdorfer on light micrographs (1250X) of toluidine-blue-stained 0.5-,I sections.
Three photographs were analyzed from each of 5 blocks for each animal. The nuclear diameter was corrected for missing small profiles by the method of Weibel and Bolender.2' The mean + SEM was calculated for organelles in stereologic analysis, and the data were analyzed statistically by the Student t test. Intragroup variation was assessed by the F ratio analysis of variance. Body weight and systolic blood pressures were measured monthly in unanesthetized rats as described previously.2
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Results
At 70 weeks of age, the body weight of WKY rats was 279 + 7 g, whereas it was 245 + 4 g in SH rats (P < 0.001). Blood pressure of SH rats at 70 weeks was elevated to hypertensive levels (199 ±8 mm Hg), as compared with that of WKY control rats (138 ± 8 mm Hg) (P < 0.001). At 95 weeks, the body weight of WKY control rats was 281 + 15 g, whereas for SH animals it was 250 ± 18 g (P < 0.001). Blood pressures of SH rats were 188 ± 3 mm Hg and 148 ± 6 mm Hg in control WKY rats (P < 0.001). These animals have been used previously for study of proteinuria and renal function.2 Adrenocortical Ultrastructure Quantitative Studies
The volume of zona reticularis cells in 95-week-old WKY rats was significantly greater than that of SH rats or of 70-week-old WKY or SH rats (Table 1). The cell volume of zona reticularis cells in SH rats at 95 weeks was comparable to that of 70-week-old animals. Mitochondrial volume/ cell and surface area/cell in WKY rats at 95 weeks were significantly greater than those in SH rats or in either group at 70 weeks of age (Tables 1 and 2). It is also of interest that the volume of mitochondria in 95-weekold SH rats is slightly less than that of 70-week-old SH rats, although the difference is not significant statistically. However, there is a highly significant decrease in surface area of SH rat mitochondrial membranes at 95 weeks of age. The volume and surface area of smooth endoplasmic reticulum in 95week-old SH and WKY rats is greater than that in 70-week-old SH or WKY rats. The zona reticularis of 95-week-old WKY rats had a larger volume of lipid droplets/cell, lysosomes, and lipofuscin (Table 1). Qualitative Changes
70 Weeks. The ultrastructure of zona reticularis cells from WKY rats was similar to that of SH rats. Sinusoids in the zona reticularis were prominent, irregular in size and shape, and often filled with red blood cells (Figure 1). The interconnection of sinusoids formed a more complex network in the zona reticularis (Figures 1 and 2) than in the zona fasciculata. Lipid droplets varied in size; a small number of parenchymal cells had enlarged lipid droplets, some of which filled a large part of the cytoplasm. Some lipid droplets were incorporated completely into mitochondria (Figure 2), and a distinct membrane derived from the mitochondrion surrounded the droplet. Lipofuscin in parenchymal cells was characterized by focal aggregates surrounded by a membrane and contained small, clear droplets interspersed in an electron-opaque matrix (Figure 2).
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A few cortical nodules extended into the zona reticularis in both WKY and SH rats. Large lipid droplets often filled a considerable portion of the cytoplasm. The lipid droplets were not surrounded by a distinct membrane, although fragments of smooth endoplasmic reticulum cisternae were observed at various points around the periphery (Figure 3). Mitochondria in cells with nodules were small in size, and considerably attenuated and possessed lamellar cristae (Figures 3 and 4). 95 Weeks. Large mitochondria occurred occasionally exclusively in zona reticularis cells of WKY at 95 weeks of age (Figure 5, inset); mitochondrial membranes rarely formed septums which segregated completely a peripheral portion of an enlarged mitochondrion. Mitochondria containing inclusions of lipid droplets were observed more frequently at 95 weeks in WKY and SH rats than at 70 weeks (Figure 6). In SH rats, mitochondrial cristae were sometimes reduced in numbers, although the number of cristae varied from one mitochondrion to another (Figure 7). Hypertrophic smooth endoplasmic reticulum was readily identified in virtually all zona reticularis cells of WKY and SH rats (Figures 5 and 7). The smooth reticulum of adrenocortical cells of SH rats was sometimes arranged in whorls composed of concentric cisternae arranged about a centrally located organelle, often a mitochondrion (Figure 7). Lipofuscin was present in focal areas of parenchymal cells of the zona reticularis and in macrophages located in the extravascular space (Figure 8). Discussion
The present study is the first report of the application of quantitative stereologic techniques to the study of adrenocortical cells in aging aniTable 2-Surface Area of Smooth Endoplasmic Reticulum and Mitochondrial Membranes/Cell (sq u) in the Zona Reticularis Cells of SH and WKY Rats Smooth Mitochondrial membranes endoplasmic reticulum Group Total * Cristae Outer and inner 70 Weeks WKY 17,392 ±898t 31,874 + 2013 22,389 ± 2062 8878 ±728 SH 16,133 ± 772 9893 ± 545 34,285 ± 1906 24,392 ± 1407 95 Weeks WKY 22,618 1151 51,326 ± 2600 37,213 ± 2043 14,113 ± 669 SH 19,877 ± 1956 6846 ± 587t 24,938 ± 2200 18,092 ± 1711 f * Inner and outer mitochondrial membanes and cristae.
t Mean ± SEM. t P < 0.001.
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mals, although these techniques have been employed widely in the study of the adrenal cortex under different experimental conditions."3,'- There have been extensive light-microscopic studies of the adrenal cortex of aged animals (reviewed by Bourne and Jayne 2). Using electron microscopy, Sato ' reported increased pigment in the inner adrenal cortex of old mice, and Bourne 27 showed unusually shaped mitochondria in the guinea pig. No significant difference between WKY rats and SH rats was seen in volume or surface area of cytoplasmic organelles in zona reticularis cells at 70 weeks of age. Our observations agree with the observations, after the use of qualitative techniques, of Tsuchiyama et al,'2 who did not observe large differences in zona fasciculata and zona reticularis, except for increased numbers of lysosomes and lipofuscin. It should be noted that our observations on the zona reticularis of 70-week-old rats in the present study differ from those on the zona fasciculata at 21 weeks of age 13; Nickerson reported that volume and surface area per cell of mitochondrial membranes and smooth endoplasmic reticulum in zona fasciculata cells of male SH rats were reduced significantly, as compared with those in WKY rats. Zona reticularis cells, however, were not examined in that study. It is therefore of interest that the zona reticularis of WKY rats differed from that of SH rats at 95 weeks of age. Between approximately 1 and 2 years of age, the reticularis cells of WKY rats became larger than those of SH rats, which were comparable in size to those of both strains at 70 weeks of age. Thus, our results point to a distinct difference in the zona reticularis that is manifested by 95 weeks of age; the precise mechanism that causes the differences is unclear, however. Genetic differences have been reported in the adrenal cortex of various strains of mice ' and may explain the results of the present study. There are no comparable studies, however, of the zona reticularis in SHR and WKY, although the zona glomerulosa of 21-week-old SH rats differs from that of WKY rats.13 Alternatively, the adrenal morphologic differences might be attributed to the hypertension itself 3 or to associated cardiovascular 3-renal 2 alterations in SH rats. A common observation in SH and WKY rats at 95 weeks was hypertrophy of smooth endoplasmic reticulum, reflected quantitatively by an increase in volume/cell and surface area/cell of this organelle. The significance of an accumulation of smooth endoplasmic reticulum is not completely clear. The smooth reticulum 'of adrenocortical cells contains enzymes for the synthesis of cholesterol and conversion of pregnenolone to progesterone ' and subsequently to 11-deoxycorticosterone.3' Nonetheless, no significant differences between old and young humans have been
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observed in the plasma levels of cortisol 32 or of basal levels of corticosterone in old and young Sprague-Dawley rats.3'3'' Tang and Phillips 3 reported recently that the adrenal cortex of old rats is capable of responding to ether stress by secretion of increased corticosterone, comparable to that of young animals 3; Hess and Riegle 33 showed decreased response to adrenocorticotropic hormone (ACTH) and ether stress in old, as compared with young, rats. There have been no functional studies, however, on the adrenal cortex in aged SH and WKY rat strains. Alterations in adrenocortical steroidogenesis of young SH rats are controversial. Freeman et al 8 reported no change in the secretion rate of corticosterone or 11-deoxycorticosterone in SH rats at 22 to 25 weeks of age. Moll et al 7 observed a suppression of 18-OH-11-deoxycorticosterone and corticosterone rates in young SH rats. Rapp and Dahl 6 showed a decreased secretion rate of deoxycorticosterone. However, control animals in the latter two studies were another Wistar strain, not the WKY animals. It is of interest that the smooth endoplasmic reticulum in SH rats is arrayed in concentric whorls, similar to that observed in the adrenal cortex of other models of hypertension studied by our laboratory.3'37 Whorls of smooth membranes occur in the adrenal cortex of rats made hypertensive with methyl-androstenediol, a synthetic androgen,3 and in the adrenal cortex of hypertensive rats bearing a mammotropic pituitary tumor secreting large quantities of ACTH, growth hormone, and prolactin.37 In these models of hypertension, there is dysfunction of the adrenal cortex resulting in an increased formation of ll-deoxycorticosterone, a known hypertensinogenic steroid,' rather than corticosterone, the principal glucocorticoid secreted by the rat adrenal gland. A reduction in 1lf,-hydroxylation and cytochrome P-450 39.40 in these models of hypertension accompanies reduced mitochondrial cristae.6'37 A significant reduction in surface area of mitochondrial cristae is seen in the zona reticularis of 95week-old SH rats in the present study. This reduction suggests the possibility that a functional change could accompany alterations in adrenocortical ultrastructure of SH rats. There is no functional data, however, on the capacity of aging SH rats to secrete steroids. It should be pointed out that it is difficult to obtain a sufficient number of SH rats for analysis of steroid levels because of difficulty in keeping female hypertensive SH rats alive for 95 weeks and beyond inasmuch as respiratory infections and changes in organs, such as the kidney, contribute to morbidity in these animals.2"7 A significant increase in volume/cell and surface area/cell of mitochondrial membranes occurs in zona reticularis cells of 95-week-old WKY animals. In small part, the presence of giant mitochondria exclusively in
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WKY rats at 95 weeks could account for increased mitochondrial volume and surface area. Hypophysectomy induces giant mitochondria in the rat adrenal cortex.4' Canick and Purivs 42 reported that the giant mitochondria reflect withdrawal of ACTH inasmuch as they are reversed following injection of exogenous ACTH. However, only a small number of giant mitochondria were seen, and there is no evidence for reduced secretion of ACTH. Septations of giant mitochondria observed in the present study may well reflect the division of mitochondria, as reported by others.42 Alternatively, we cannot exclude the possibility that they represent the fusion of other mitochondria with larger structures. The incorporation of lipid droplets within mitochondria in zona reticularis cells of WKY and SH rats could reflect a degenerative change in the cells. The association of lipid droplets with mitochondria has been reported previously by our laboratory in zona reticularis cells of retired breeder gerbils and hamsters.'6 Recently, we have also described similar changes in zona reticularis cells of rats after thyroparathyroidectomy.43 Decreased thyroid activity has been reported in young mature SH rats,," although recently normal to elevated T3 levels have also been shown.45 It is unlikely that changes in thyroid function were involved in induction of mitochondria-lipid-droplet associations, because these structures were present in both SH and WKY rats. The inclusions may reflect a reduced capacity for cholesterol metabolism inasmuch as identical types of inclusions have been reported after administration of aminoglutethimide,4 a drug which inhibits the conversion of cholesterol to pregnenolone. Increase numbers of lipid droplets that contain predominantly cholesterol and cholesterol esters 47 could then accumulate within mitochondria. The ultrastructure of cortical nodules in the present study was virtually identical to that described by Sugihara et al.48 Cortical nodules are found frequently within the adrenal cortex of aged animals.49 References 1. Okamoto K, Aoki K: Development of a strain of spontaneously hypertensive rats. Jpn Circ J 27:282-293, 1963 2. Feld LG, VanLiew JB, Galaske RG, Boylan JW: Selectivity of renal injury and proteinuria in the spontaneously hypertensive rat. Kidney Int 12:332-343, 1977 3. Okamoto K, Aoki K, Nosaka S, Fukushima W: Cardiovascular diseases in the spontaneously hypertensive rat. Jpn Circ J 28:943-952, 1964 4. Aoki K, Tankawa H, Fujinami T, Miyazaki A, Hashimoto Y: Pathological studies on the endocrine organs of the spontaneously hypertensive rats. Jpn Heart J 4:426-442, 1963 5. Aoki K: Experimental studies on the relationship between endocrine organs and hypertension in spontaneously hypertensive rats: I. Effects of hypophysectomy, adrenalectomy, thyroidectomy and sympathectomy on blood pressure. Jpn Heart J 4:443-461, 1963
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6. Rapp JP, Dahl LK: Adrenal steroidogenesis in rats bred for susceptibility and resistance to the hypertensive effect of salt. Endocrinology 88:52-65, 1971 7. Moll D, Dale SL, Melby JC: Adrenal steroidogenesis in the spontaneously hypertensive rat (SHR). Endocrinology 96:416-420, 1975 8. Freeman RH, Davis JO, Varsano-Aharon N, Ulick S, Weinberger MH: Control of aldosterone secretion in the spontaneously hypertensive rat. Circ Res 37:66-71, 1975 9. Fregly MJ: Thyroid activity of spontaneous hypertensive rats. Proc Soc Exp Biol Med 149:124-132, 1975 10. Baer L, Knowlton A, Laragh JH: The role of sodium balance and the pituitary adrenal axis in the hypertension of spontaneously hypertensive rats, Spontaneous Hypertension: Its Pathogenesis and Complications. Edited by K Okamoto. Tokyo, Igaku Shoin Ltd., 1972, pp 203-209 11. Maruyama T: Electron microscopic studies on the adrenal medulla and adrenal cortex of hypertensive rats: I. Spontaneously hypertensive rats. Jpn Circ J 33:12711284, 1969 12. Tsuchiyama H, Sugihara H, Kawai K: Pathology of the adrenal cortex in spontaneously hypertensive rats,'0 pp 177-184 13. Nickerson PA: The adrenal cortex in spontaneously hypertensive rats: A quantitative ultrastructural study. Am J Pathol 84:545-560, 1976 14. Bartsch G, Baumgartner U, Rohr HP: A stereological study of adrenocortical cells in spontaneously hypertensive rats (SHR). Path Res Pract 162:291-300, 1978 15. Hirano T: Histochemical and ultrastructural studies of the adrenal cortex in spontaneously hypertensive rats with high salt solution. Acta Pathol Jpn 26:589-601, 1976 16. Nickerson PA: Adrenal cortex in retired breeder Mongolian gerbils (Meriones unguiculatus) and golden hamsters (Mesocricetus auratus): Ultrastructural alterations in the zona reticularis. Am J Pathol 95:347-358, 1979 17. Udenfriend S, Bumpus FM, Foster HL, Freis ED, Hansen CT, Lovenberg WM, Yamori Y: Spontaneously hypertensive (SHR) rats: Guidelines for breeding, care, and use. Inst Lab Anim Res News 19(3):G3-G20, 1976 18. Nickerson PA, Brownie AC, Skelton FR: An electron microscopic study of the regeneration adrenal gland during the development of adrenal regeneration hypertension. Am J Pathol 57:335-364, 1969 19. Stempak JG, Ward RT: An improved staining method for electron microscopy. J Cell Biol 22:697-701, 1964 20. Reynolds ES: The use of lead citrate at high pH as an electron-opaque stain in elec tron microscopy. J Cell Biol 17:208-212, 1963 21. Weibel ER, Bolender RP: Stereological techniques for electron microscopic morphometry, Principles and Techniques of Electron Microscopy. Vol 3. Edited by MA Hayat. New York, Van Nostrand Reinhold Co., 1973, pp 237-296 22. Nussdorfer GG: Effects of corticosteroid-hormones on the smooth endoplasmic reticulum of rat adrenocortical cells. Z Zellforsch Mikrosk Anat 106:143-154, 1970 23. Kasemsri S, Nickerson PA: Quantitative ultrastructural study of the rat adrenal cortex in renal encapsulation-induced hypertension. Am J Pathol 82:143-156, 1976 24. Nickerson PA: A quantitative ultrastructural study of the adrenal gland in rats resistant and sensitive to the hypertensive effects of sodium chloride. Lab Invest 37:120-125, 1977 25. Boume GH, Jayne EP: The adrenal gland, Structural Aspects of Aging. Edited by GH Boume. New York, Hafner Publ Co., 1961, pp 305-324 26. Sato T: Age and sex differences in the fine structure of the mouse adrenal cortex. Nagoya J Med Sci 30:225-251, 1967 27. Boume GH: Aging changes in the endocrines, Endocrines and Aging. Chap 4. Edited by L Gitman. Springfield, Ill, Charles C Thomas, 1967, pp 66-75
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28. Shire JGM, Spickett SG: Genetic variation in adrenal structure: Qualitative differences in the zona glomerulosa. J Endocrinol 39:277-284, 1967 29. Chesterton CJ: Distribution of cholesterol precursors and other lipids among rat liver intracellular structures: Evidence for the endoplasmic reticulum as the site of cholesterol and cholesterol ester synthesis. J Biol Chem 243:1147-1151, 1968 30. Beyer KF, Samuels LT: Distribution of steroid-3f8-ol-dehydrogenase in cellular structures of the adrenal gland. J Biol Chem 219:69-76, 1956 31. Ryan KJ, Engel LL: Hydroxylation of steroid at carbon 21. J Biol Chem 225:103114, 1957 32. Jensen HK, Blichert-Toft M: Serum corticotrophin, plasma cortisol, and urinary excretion of 17-ketogenic steroids in the elderly (age group: 66-94 years) Acta Endocrinol 66:25-34, 1971 33. Hess GD, Riegle GD: Adrenocortical responsiveness to stress and ACTH in aging rats. J Gerontol 25:354-358, 1970 34. Hess GD, Riegle GD: Effects of chronic ACTH stimulation on adrenocortical function in young and aged rats. Am J Physiol 222:1458-1461, 1972 35. Tang F, Phillips JG: Some age-related changes in pituitary-adrenal function in the male laboratory rat. J Gerontol 33:377-382, 1978 36. Skelton, FR, Brownie AC: Studies on the pathogenesis of adrenal-regeneration and methylandrostenediol hypertension, Endocrine Aspects of Disease Process. Edited by G Jasmin. St. Louis, Warren H. Green Inc., 1968, pp 271-301 37. Nickerson PA, Brownie AC, Molteni A: Adrenocortical structure and function in rats bearing an adrenocorticotrophic hormone, growth hormone, and prolactin-secreting tumor. Lab Invest 23:368-377, 1970 38. Selye H, Hall CE, Rowley EM: Malignant hypertension produced by treatment with desoxycorticosterone acetate and sodium chloride. Can Med Assoc J 49:88-92, 1943 39. Brownie AC, Simpson ER, Skelton FR, Elliott WB, Estabrook RW: Interaction of androgens with the adrenal mitochondrial cytochrome system: The influence of androgen treatment on the levels of cytochrome in rat adrenal cortical mitochondria and on substrate-induced spectral changes. Arch Biochem Biophys 141:18-25, 1970 40. Brownie AC, Colby HD, Gallant S, Skelton FR: Some studies on the effect of androgens on adrenal cortical function of rats. Endocrinology 86:1085-1092, 1970 41. Volk TL, Scarpelli DG: Mitochondrial gigantism in the adrenal cortex following hypophysectomy. Lab Invest 15:707-715, 1966 42. Canick JA, Purvis JL: The maintenance of mitochondrial size in the rat adrenal cortex zona fasciculata by ACTH. Exp Molec Pathol 16:79-93, 1972 43. Conran RM, Nickerson PA: Effect of thyroparathyroidectomy (TPX) on the zona reticularis: A quantitative ultrastructural study. Anat Rec 194:405-416, 1979 44. Fregly MJ: Thyroid activity of spontaneous hypertensive rats. Proc Soc Exp Biol Med 149:124-132, 1975 45. Wright GL, Knecht E, Badger D, Samueloff S, Toraason M, Dukes-Dobas F: Oxygen consumption in the spontaneously hypertensive rat. Proc Soc Exp Biol Med 159:449-452, 1978 46. Marek J, Thoenes W, Motlik K: Lipoide Transformation der Mitochondrien in Nebennierenrindenzellen nach Aminoglutathimid (Elipten Ciba). Virchows Archiv [Cell Pathol] 6:116-131, 1970 47. Malamed S: Ultrastructure of the mammalian adrenal cortex in relation to secretory function, Handbook of Physiology. Section 7, Vol 6, Adrenal Gland. Edited by RO Greep, EB Astwood. American Physiological Society, Washington, DC, 1975, pp 25-39
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48. Sugihara H, Kawai K, Tsuchiyama H: Pathology of intracortical nodules in rat adrenal glands, especially on their fine-structure. Acta Pathol Jpn 23:253-260, 1973 49. Dobbie JW: Adrenocortical nodular hyperplasia: The ageing adrenal. J Pathol 99:1-18, 1969
Acknowledgments Mrs. Neonila Fylypiw, Mrs. Priscilla Adams, Mrs. Elizabeth Lawson, Mrs. Geneva Joseph, Mrs. Luise Bohacek, and Mr. Robert Linsmair provided skilled technical assistance. Mrs. Berta Cole typed the manuscript.
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[Illustrations follow]
Figure 1-Zona reticularis cell from an SH rat 70 weeks of age. sinusoids are filled with red blood cells (R). Parenchymal cells contain lipid droplets inProminent the cytoplasm readily identified by their electron-lucid matrix. (x5520) Figure 2-Zona reticularis cell from a WKY rat 70 weeks of age. A large single lipid droplet (L) is incorporated into a Mitochondrial cristae (C) are observed at one point; mitochondrial members aremitochondrion. double and attenuated, and contain no cristae at another point (arrows). Several accumulations of lipofuscin (F) are observed in the cytoplasm. Smooth endoplasmic reticulum (ER). (x 18,400) Inset-4n a 0.5-u toluidine-blue-stained section, some cells contain enlarged, clear vacuolar lipid droplets. (x725) (Both with a photographic reduction of 5%)
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of age. A Figure 3-Cell from a nodule within the zona reticularis of a WKY rat at 70 weeks limiting membrane, alsingle lipid droplet (L) occupies almost the entire cytoplasm. There is no of though discontinuous cisternae of smooth endoplasmic reticulum are seen at the periphery Figure 4-Cell from a the droplet (arrows). Mitochondrial cristae are lamellar. (xl 1,600) nodule within the zona reticularis of a WKY rat at 70 weeks of age. Mitochondria in some parenchymal cells are attenuated and contain lamellar cristae. (x1 2,600) (Both with a photographic reduction of 5%)
Figure 5-Zona reticularis from a WKY rat 95 weeks of age. Smooth endoplasmic reticulum (ER) is hypertrophic. Lipofuscin (F). (xi 6,000) Inset-An especially large A septum (arrow) isolates a peripheral area of the mitochrondrion. (X10,400) mitochondrion. Figure 6Zona reticularis cell from a WKY rat 95 weeks of age. A single large lipid droplet is encircled a mitochondrion. At one point, there is apparently a septation (arrow) of the mitochondria by surrounding the lipid droplet. (x 16,000) (Both with a photographic reduction of 5%)
Figure 7-Zona reticularis from a SH rat at 95 weeks of age. Hypertrophic smooth endoplasmic reticulum (ER) is arranged in concentric lamellar formations. Mitochondria contain small numFigure 8-Macrophage in the inner zona reticubers of tubulovesicular cristae. (x 17,600) laris adjacent to the medulla of a SH rat 95 weeks of age. The macrophage can be identified by small mitochondria with lamellar cristae. Discrete areas of lipofuscin (F) and membrane-bound vacuoles (V) with a moderately electron-opaque matrix can be seen throughout the cytoplasm. Membrane-bound granules with an electron-opaque core are present in a medullary cell (M). (x 17,600) (Both with a photographic reduction of 5%)
The zona reticularis of 70- and 95-week-old female Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats was studied by quantitative stereologic techniques. The zona reticularis in the adrenal gland of 70-week-old SH rats was virtually identical to that of 70-week-old W7KY rats. In both SH and WKY rats at 95 weeks of age, however, there was hypertrophy of smooth endoplasmic reticulum reflected quantitatively in increased volume and surface area, as compared with that of WKY rats or SH rats at 70 weeks of age. Ninety-five-week-old WVKY rats had a significantly greater mitochondrial volume/cell and surface area of mitochondrial membranes than SH rats. Inclusion of lipid droplets within mitochondria was seen in zona reticularis cells from SH and WKY rats at 70 weeks of age; mitochondria-lipid-droplet association was more frequent at 95 weeks of age. The volume of lipofuscin per cell was significantly greater in WVKY than in SH rats at 95 weeks of age. Thus, by quantitative techniques, one can see that the zona reticularis of 95-week-old SH rats differs from that of WKY rats, principally in the presence of smaller cells with a smaller surface area of mitochondrial cristae and a reduced volume of mitochondria, lipofuscin, and lipid droplets. (Am J Pathol 97:433-448, 1979)
OKAMOTO AND AOKI 1 selectively bred Wistar-Kyoto (WKY) rats to form a strain of rats that spontaneously develop hypertension (SH). SH rats develop hypertension,l cardiomegaly, nephrosclerosis,2 vascular disease, and cerebral hemorrhage 3 without any experimental treatment. Abnormalities of the pituitary-adrenal axis,4'5 steroid synthesis, 8 and the thyroid gland9 have been implicated in the pathogenesis of the hypertension, although the essentiality of the endocrine glands in development of the cardiovascular disease has not been proven conclusively.'0 There have been several studies of adrenocortical ultrastructure in SH rats."1-5 Nickerson 13 has shown by quantitative morphologic techniques that the zona glomerulosa and zona fasciculata of 21-week-old male SH rats differ from those of WKY animals. Quantitative morphologic techniques also emphasized the essentiality of using the WKY rat as a control From the Department of Pathology, State University of New York at Buffalo, and the Department of Physiology, Veterans Administration Medical Center, Buffalo, New York. Supported by research grants from the National Heart, Lung and Blood Institute (HL-06975) and the American Heart Association and Veterans Administration Research Funds. Accepted for publication June 29, 1979. Address reprint requests to Peter A. Nickerson, PhD, Department of Pathology, State University of New York at Buffalo, 204 Farber Hall, Buffalo, NY 14214. 0002-9440/79/11 08-0433$01 .00 433 © American Association of Pathologists
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because the adrenal cortex of WKY differs considerably from that of another strain of Wistar rat available in the United States.'3 Most recently, Bartsch et al 14 reported increases in surface area and volume of smooth endoplasmic reticulum in the zona fasciculata from 7-month-old SH rats by stereologic techniques; however, they did not employ WKY rats as controls. Tsuchiyama et al 12 have also examined the adrenal gland of the SH rat and observed an increased number of lysosomes and lipofuchsin pigment in the inner zona fasciculata and zona reticularis in 12-16month-old male rats. The adrenal glands of aging rats have not been studied extensively by electron microscopy. Recently, Nickerson 16 examined the adrenal cortex of retired breeder gerbils and hamsters and reported alterations in the zona reticularis. We therefore wanted to investigate further the zona reticularis in aging WKY and SH rats by quantitative stereologic techniques to assess morphologic alterations by an objective method. Materials and Methods Okamoto SH and WKY female rats were obtained from Laboratory Supply Company, Indianapolis, and raised in animal holding facilities at the Veterans Administration Medical Center. SH and WKY rats were F-generation 42 and 19, respectively. Animals were caged individually and given lab chow and tap water ad libitum. Access to the facility was limited in order to minimize the possibility of introducing infection to the animals.'7 Three rats each of the WKY and SH strains, 70 and 95 weeks of age, were anesthetized with Inactin (Byk Gulden Konstanz). The adrenal gland was removed quickly and trimmed of adherent fat; 1-mm slices were cut and fixed in 3% purified glutaraldehyde (Ladd Research Industries, Burlington, Vt) buffered to pH 7.3 with 0.1 M phosphate. Tissues were washed in 0.1 M phosphate buffer (pH 7.4). While in buffer, radial sections were cut with a microscalpel while at 3x magnification in a dissecting microscope. The sections were cut so as to include a small piece of adrenal medulla, which facilitated the selection and proper orientation of the tissue. Several blocks with hemorrhagic areas and poorly fixed cells were identified by light microscopy and not included in the analysis. Tissues were processed for electron microscopy as described previously.'8 Half-micron-thick sections were cut from tissue blocks, stained with toluidine blue, and examined by light microscopy to confirm the zonal position of the tissue. Thin sections were cut with glass knives on a Porter-Blum MT-1 ultramicrotome, stained with uranyl acetate '" and lead citrate,20 and examined with a Siemens 101 electron microscope. For quantitative techniques, 4 photographs were taken at low (2400X) and high magnification (8000X) from each of 5 tissue blocks for each animal and magnified 3.5X photographically. Magnification was calibrated with a replica grating (Ernest Fullam, Schenectady, NY). Volume and surface area measurements were made on photographic prints according to the method of Weibel and Bolender 2' and had been used previously by our
laboratory.'3 The average volume per adrenocortical cell was determined by the method of Nussdorfer on light micrographs (1250X) of toluidine-blue-stained 0.5-,I sections.
Three photographs were analyzed from each of 5 blocks for each animal. The nuclear diameter was corrected for missing small profiles by the method of Weibel and Bolender.2' The mean + SEM was calculated for organelles in stereologic analysis, and the data were analyzed statistically by the Student t test. Intragroup variation was assessed by the F ratio analysis of variance. Body weight and systolic blood pressures were measured monthly in unanesthetized rats as described previously.2
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Results
At 70 weeks of age, the body weight of WKY rats was 279 + 7 g, whereas it was 245 + 4 g in SH rats (P < 0.001). Blood pressure of SH rats at 70 weeks was elevated to hypertensive levels (199 ±8 mm Hg), as compared with that of WKY control rats (138 ± 8 mm Hg) (P < 0.001). At 95 weeks, the body weight of WKY control rats was 281 + 15 g, whereas for SH animals it was 250 ± 18 g (P < 0.001). Blood pressures of SH rats were 188 ± 3 mm Hg and 148 ± 6 mm Hg in control WKY rats (P < 0.001). These animals have been used previously for study of proteinuria and renal function.2 Adrenocortical Ultrastructure Quantitative Studies
The volume of zona reticularis cells in 95-week-old WKY rats was significantly greater than that of SH rats or of 70-week-old WKY or SH rats (Table 1). The cell volume of zona reticularis cells in SH rats at 95 weeks was comparable to that of 70-week-old animals. Mitochondrial volume/ cell and surface area/cell in WKY rats at 95 weeks were significantly greater than those in SH rats or in either group at 70 weeks of age (Tables 1 and 2). It is also of interest that the volume of mitochondria in 95-weekold SH rats is slightly less than that of 70-week-old SH rats, although the difference is not significant statistically. However, there is a highly significant decrease in surface area of SH rat mitochondrial membranes at 95 weeks of age. The volume and surface area of smooth endoplasmic reticulum in 95week-old SH and WKY rats is greater than that in 70-week-old SH or WKY rats. The zona reticularis of 95-week-old WKY rats had a larger volume of lipid droplets/cell, lysosomes, and lipofuscin (Table 1). Qualitative Changes
70 Weeks. The ultrastructure of zona reticularis cells from WKY rats was similar to that of SH rats. Sinusoids in the zona reticularis were prominent, irregular in size and shape, and often filled with red blood cells (Figure 1). The interconnection of sinusoids formed a more complex network in the zona reticularis (Figures 1 and 2) than in the zona fasciculata. Lipid droplets varied in size; a small number of parenchymal cells had enlarged lipid droplets, some of which filled a large part of the cytoplasm. Some lipid droplets were incorporated completely into mitochondria (Figure 2), and a distinct membrane derived from the mitochondrion surrounded the droplet. Lipofuscin in parenchymal cells was characterized by focal aggregates surrounded by a membrane and contained small, clear droplets interspersed in an electron-opaque matrix (Figure 2).
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A few cortical nodules extended into the zona reticularis in both WKY and SH rats. Large lipid droplets often filled a considerable portion of the cytoplasm. The lipid droplets were not surrounded by a distinct membrane, although fragments of smooth endoplasmic reticulum cisternae were observed at various points around the periphery (Figure 3). Mitochondria in cells with nodules were small in size, and considerably attenuated and possessed lamellar cristae (Figures 3 and 4). 95 Weeks. Large mitochondria occurred occasionally exclusively in zona reticularis cells of WKY at 95 weeks of age (Figure 5, inset); mitochondrial membranes rarely formed septums which segregated completely a peripheral portion of an enlarged mitochondrion. Mitochondria containing inclusions of lipid droplets were observed more frequently at 95 weeks in WKY and SH rats than at 70 weeks (Figure 6). In SH rats, mitochondrial cristae were sometimes reduced in numbers, although the number of cristae varied from one mitochondrion to another (Figure 7). Hypertrophic smooth endoplasmic reticulum was readily identified in virtually all zona reticularis cells of WKY and SH rats (Figures 5 and 7). The smooth reticulum of adrenocortical cells of SH rats was sometimes arranged in whorls composed of concentric cisternae arranged about a centrally located organelle, often a mitochondrion (Figure 7). Lipofuscin was present in focal areas of parenchymal cells of the zona reticularis and in macrophages located in the extravascular space (Figure 8). Discussion
The present study is the first report of the application of quantitative stereologic techniques to the study of adrenocortical cells in aging aniTable 2-Surface Area of Smooth Endoplasmic Reticulum and Mitochondrial Membranes/Cell (sq u) in the Zona Reticularis Cells of SH and WKY Rats Smooth Mitochondrial membranes endoplasmic reticulum Group Total * Cristae Outer and inner 70 Weeks WKY 17,392 ±898t 31,874 + 2013 22,389 ± 2062 8878 ±728 SH 16,133 ± 772 9893 ± 545 34,285 ± 1906 24,392 ± 1407 95 Weeks WKY 22,618 1151 51,326 ± 2600 37,213 ± 2043 14,113 ± 669 SH 19,877 ± 1956 6846 ± 587t 24,938 ± 2200 18,092 ± 1711 f * Inner and outer mitochondrial membanes and cristae.
t Mean ± SEM. t P < 0.001.
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mals, although these techniques have been employed widely in the study of the adrenal cortex under different experimental conditions."3,'- There have been extensive light-microscopic studies of the adrenal cortex of aged animals (reviewed by Bourne and Jayne 2). Using electron microscopy, Sato ' reported increased pigment in the inner adrenal cortex of old mice, and Bourne 27 showed unusually shaped mitochondria in the guinea pig. No significant difference between WKY rats and SH rats was seen in volume or surface area of cytoplasmic organelles in zona reticularis cells at 70 weeks of age. Our observations agree with the observations, after the use of qualitative techniques, of Tsuchiyama et al,'2 who did not observe large differences in zona fasciculata and zona reticularis, except for increased numbers of lysosomes and lipofuscin. It should be noted that our observations on the zona reticularis of 70-week-old rats in the present study differ from those on the zona fasciculata at 21 weeks of age 13; Nickerson reported that volume and surface area per cell of mitochondrial membranes and smooth endoplasmic reticulum in zona fasciculata cells of male SH rats were reduced significantly, as compared with those in WKY rats. Zona reticularis cells, however, were not examined in that study. It is therefore of interest that the zona reticularis of WKY rats differed from that of SH rats at 95 weeks of age. Between approximately 1 and 2 years of age, the reticularis cells of WKY rats became larger than those of SH rats, which were comparable in size to those of both strains at 70 weeks of age. Thus, our results point to a distinct difference in the zona reticularis that is manifested by 95 weeks of age; the precise mechanism that causes the differences is unclear, however. Genetic differences have been reported in the adrenal cortex of various strains of mice ' and may explain the results of the present study. There are no comparable studies, however, of the zona reticularis in SHR and WKY, although the zona glomerulosa of 21-week-old SH rats differs from that of WKY rats.13 Alternatively, the adrenal morphologic differences might be attributed to the hypertension itself 3 or to associated cardiovascular 3-renal 2 alterations in SH rats. A common observation in SH and WKY rats at 95 weeks was hypertrophy of smooth endoplasmic reticulum, reflected quantitatively by an increase in volume/cell and surface area/cell of this organelle. The significance of an accumulation of smooth endoplasmic reticulum is not completely clear. The smooth reticulum 'of adrenocortical cells contains enzymes for the synthesis of cholesterol and conversion of pregnenolone to progesterone ' and subsequently to 11-deoxycorticosterone.3' Nonetheless, no significant differences between old and young humans have been
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observed in the plasma levels of cortisol 32 or of basal levels of corticosterone in old and young Sprague-Dawley rats.3'3'' Tang and Phillips 3 reported recently that the adrenal cortex of old rats is capable of responding to ether stress by secretion of increased corticosterone, comparable to that of young animals 3; Hess and Riegle 33 showed decreased response to adrenocorticotropic hormone (ACTH) and ether stress in old, as compared with young, rats. There have been no functional studies, however, on the adrenal cortex in aged SH and WKY rat strains. Alterations in adrenocortical steroidogenesis of young SH rats are controversial. Freeman et al 8 reported no change in the secretion rate of corticosterone or 11-deoxycorticosterone in SH rats at 22 to 25 weeks of age. Moll et al 7 observed a suppression of 18-OH-11-deoxycorticosterone and corticosterone rates in young SH rats. Rapp and Dahl 6 showed a decreased secretion rate of deoxycorticosterone. However, control animals in the latter two studies were another Wistar strain, not the WKY animals. It is of interest that the smooth endoplasmic reticulum in SH rats is arrayed in concentric whorls, similar to that observed in the adrenal cortex of other models of hypertension studied by our laboratory.3'37 Whorls of smooth membranes occur in the adrenal cortex of rats made hypertensive with methyl-androstenediol, a synthetic androgen,3 and in the adrenal cortex of hypertensive rats bearing a mammotropic pituitary tumor secreting large quantities of ACTH, growth hormone, and prolactin.37 In these models of hypertension, there is dysfunction of the adrenal cortex resulting in an increased formation of ll-deoxycorticosterone, a known hypertensinogenic steroid,' rather than corticosterone, the principal glucocorticoid secreted by the rat adrenal gland. A reduction in 1lf,-hydroxylation and cytochrome P-450 39.40 in these models of hypertension accompanies reduced mitochondrial cristae.6'37 A significant reduction in surface area of mitochondrial cristae is seen in the zona reticularis of 95week-old SH rats in the present study. This reduction suggests the possibility that a functional change could accompany alterations in adrenocortical ultrastructure of SH rats. There is no functional data, however, on the capacity of aging SH rats to secrete steroids. It should be pointed out that it is difficult to obtain a sufficient number of SH rats for analysis of steroid levels because of difficulty in keeping female hypertensive SH rats alive for 95 weeks and beyond inasmuch as respiratory infections and changes in organs, such as the kidney, contribute to morbidity in these animals.2"7 A significant increase in volume/cell and surface area/cell of mitochondrial membranes occurs in zona reticularis cells of 95-week-old WKY animals. In small part, the presence of giant mitochondria exclusively in
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WKY rats at 95 weeks could account for increased mitochondrial volume and surface area. Hypophysectomy induces giant mitochondria in the rat adrenal cortex.4' Canick and Purivs 42 reported that the giant mitochondria reflect withdrawal of ACTH inasmuch as they are reversed following injection of exogenous ACTH. However, only a small number of giant mitochondria were seen, and there is no evidence for reduced secretion of ACTH. Septations of giant mitochondria observed in the present study may well reflect the division of mitochondria, as reported by others.42 Alternatively, we cannot exclude the possibility that they represent the fusion of other mitochondria with larger structures. The incorporation of lipid droplets within mitochondria in zona reticularis cells of WKY and SH rats could reflect a degenerative change in the cells. The association of lipid droplets with mitochondria has been reported previously by our laboratory in zona reticularis cells of retired breeder gerbils and hamsters.'6 Recently, we have also described similar changes in zona reticularis cells of rats after thyroparathyroidectomy.43 Decreased thyroid activity has been reported in young mature SH rats,," although recently normal to elevated T3 levels have also been shown.45 It is unlikely that changes in thyroid function were involved in induction of mitochondria-lipid-droplet associations, because these structures were present in both SH and WKY rats. The inclusions may reflect a reduced capacity for cholesterol metabolism inasmuch as identical types of inclusions have been reported after administration of aminoglutethimide,4 a drug which inhibits the conversion of cholesterol to pregnenolone. Increase numbers of lipid droplets that contain predominantly cholesterol and cholesterol esters 47 could then accumulate within mitochondria. The ultrastructure of cortical nodules in the present study was virtually identical to that described by Sugihara et al.48 Cortical nodules are found frequently within the adrenal cortex of aged animals.49 References 1. Okamoto K, Aoki K: Development of a strain of spontaneously hypertensive rats. Jpn Circ J 27:282-293, 1963 2. Feld LG, VanLiew JB, Galaske RG, Boylan JW: Selectivity of renal injury and proteinuria in the spontaneously hypertensive rat. Kidney Int 12:332-343, 1977 3. Okamoto K, Aoki K, Nosaka S, Fukushima W: Cardiovascular diseases in the spontaneously hypertensive rat. Jpn Circ J 28:943-952, 1964 4. Aoki K, Tankawa H, Fujinami T, Miyazaki A, Hashimoto Y: Pathological studies on the endocrine organs of the spontaneously hypertensive rats. Jpn Heart J 4:426-442, 1963 5. Aoki K: Experimental studies on the relationship between endocrine organs and hypertension in spontaneously hypertensive rats: I. Effects of hypophysectomy, adrenalectomy, thyroidectomy and sympathectomy on blood pressure. Jpn Heart J 4:443-461, 1963
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6. Rapp JP, Dahl LK: Adrenal steroidogenesis in rats bred for susceptibility and resistance to the hypertensive effect of salt. Endocrinology 88:52-65, 1971 7. Moll D, Dale SL, Melby JC: Adrenal steroidogenesis in the spontaneously hypertensive rat (SHR). Endocrinology 96:416-420, 1975 8. Freeman RH, Davis JO, Varsano-Aharon N, Ulick S, Weinberger MH: Control of aldosterone secretion in the spontaneously hypertensive rat. Circ Res 37:66-71, 1975 9. Fregly MJ: Thyroid activity of spontaneous hypertensive rats. Proc Soc Exp Biol Med 149:124-132, 1975 10. Baer L, Knowlton A, Laragh JH: The role of sodium balance and the pituitary adrenal axis in the hypertension of spontaneously hypertensive rats, Spontaneous Hypertension: Its Pathogenesis and Complications. Edited by K Okamoto. Tokyo, Igaku Shoin Ltd., 1972, pp 203-209 11. Maruyama T: Electron microscopic studies on the adrenal medulla and adrenal cortex of hypertensive rats: I. Spontaneously hypertensive rats. Jpn Circ J 33:12711284, 1969 12. Tsuchiyama H, Sugihara H, Kawai K: Pathology of the adrenal cortex in spontaneously hypertensive rats,'0 pp 177-184 13. Nickerson PA: The adrenal cortex in spontaneously hypertensive rats: A quantitative ultrastructural study. Am J Pathol 84:545-560, 1976 14. Bartsch G, Baumgartner U, Rohr HP: A stereological study of adrenocortical cells in spontaneously hypertensive rats (SHR). Path Res Pract 162:291-300, 1978 15. Hirano T: Histochemical and ultrastructural studies of the adrenal cortex in spontaneously hypertensive rats with high salt solution. Acta Pathol Jpn 26:589-601, 1976 16. Nickerson PA: Adrenal cortex in retired breeder Mongolian gerbils (Meriones unguiculatus) and golden hamsters (Mesocricetus auratus): Ultrastructural alterations in the zona reticularis. Am J Pathol 95:347-358, 1979 17. Udenfriend S, Bumpus FM, Foster HL, Freis ED, Hansen CT, Lovenberg WM, Yamori Y: Spontaneously hypertensive (SHR) rats: Guidelines for breeding, care, and use. Inst Lab Anim Res News 19(3):G3-G20, 1976 18. Nickerson PA, Brownie AC, Skelton FR: An electron microscopic study of the regeneration adrenal gland during the development of adrenal regeneration hypertension. Am J Pathol 57:335-364, 1969 19. Stempak JG, Ward RT: An improved staining method for electron microscopy. J Cell Biol 22:697-701, 1964 20. Reynolds ES: The use of lead citrate at high pH as an electron-opaque stain in elec tron microscopy. J Cell Biol 17:208-212, 1963 21. Weibel ER, Bolender RP: Stereological techniques for electron microscopic morphometry, Principles and Techniques of Electron Microscopy. Vol 3. Edited by MA Hayat. New York, Van Nostrand Reinhold Co., 1973, pp 237-296 22. Nussdorfer GG: Effects of corticosteroid-hormones on the smooth endoplasmic reticulum of rat adrenocortical cells. Z Zellforsch Mikrosk Anat 106:143-154, 1970 23. Kasemsri S, Nickerson PA: Quantitative ultrastructural study of the rat adrenal cortex in renal encapsulation-induced hypertension. Am J Pathol 82:143-156, 1976 24. Nickerson PA: A quantitative ultrastructural study of the adrenal gland in rats resistant and sensitive to the hypertensive effects of sodium chloride. Lab Invest 37:120-125, 1977 25. Boume GH, Jayne EP: The adrenal gland, Structural Aspects of Aging. Edited by GH Boume. New York, Hafner Publ Co., 1961, pp 305-324 26. Sato T: Age and sex differences in the fine structure of the mouse adrenal cortex. Nagoya J Med Sci 30:225-251, 1967 27. Boume GH: Aging changes in the endocrines, Endocrines and Aging. Chap 4. Edited by L Gitman. Springfield, Ill, Charles C Thomas, 1967, pp 66-75
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28. Shire JGM, Spickett SG: Genetic variation in adrenal structure: Qualitative differences in the zona glomerulosa. J Endocrinol 39:277-284, 1967 29. Chesterton CJ: Distribution of cholesterol precursors and other lipids among rat liver intracellular structures: Evidence for the endoplasmic reticulum as the site of cholesterol and cholesterol ester synthesis. J Biol Chem 243:1147-1151, 1968 30. Beyer KF, Samuels LT: Distribution of steroid-3f8-ol-dehydrogenase in cellular structures of the adrenal gland. J Biol Chem 219:69-76, 1956 31. Ryan KJ, Engel LL: Hydroxylation of steroid at carbon 21. J Biol Chem 225:103114, 1957 32. Jensen HK, Blichert-Toft M: Serum corticotrophin, plasma cortisol, and urinary excretion of 17-ketogenic steroids in the elderly (age group: 66-94 years) Acta Endocrinol 66:25-34, 1971 33. Hess GD, Riegle GD: Adrenocortical responsiveness to stress and ACTH in aging rats. J Gerontol 25:354-358, 1970 34. Hess GD, Riegle GD: Effects of chronic ACTH stimulation on adrenocortical function in young and aged rats. Am J Physiol 222:1458-1461, 1972 35. Tang F, Phillips JG: Some age-related changes in pituitary-adrenal function in the male laboratory rat. J Gerontol 33:377-382, 1978 36. Skelton, FR, Brownie AC: Studies on the pathogenesis of adrenal-regeneration and methylandrostenediol hypertension, Endocrine Aspects of Disease Process. Edited by G Jasmin. St. Louis, Warren H. Green Inc., 1968, pp 271-301 37. Nickerson PA, Brownie AC, Molteni A: Adrenocortical structure and function in rats bearing an adrenocorticotrophic hormone, growth hormone, and prolactin-secreting tumor. Lab Invest 23:368-377, 1970 38. Selye H, Hall CE, Rowley EM: Malignant hypertension produced by treatment with desoxycorticosterone acetate and sodium chloride. Can Med Assoc J 49:88-92, 1943 39. Brownie AC, Simpson ER, Skelton FR, Elliott WB, Estabrook RW: Interaction of androgens with the adrenal mitochondrial cytochrome system: The influence of androgen treatment on the levels of cytochrome in rat adrenal cortical mitochondria and on substrate-induced spectral changes. Arch Biochem Biophys 141:18-25, 1970 40. Brownie AC, Colby HD, Gallant S, Skelton FR: Some studies on the effect of androgens on adrenal cortical function of rats. Endocrinology 86:1085-1092, 1970 41. Volk TL, Scarpelli DG: Mitochondrial gigantism in the adrenal cortex following hypophysectomy. Lab Invest 15:707-715, 1966 42. Canick JA, Purvis JL: The maintenance of mitochondrial size in the rat adrenal cortex zona fasciculata by ACTH. Exp Molec Pathol 16:79-93, 1972 43. Conran RM, Nickerson PA: Effect of thyroparathyroidectomy (TPX) on the zona reticularis: A quantitative ultrastructural study. Anat Rec 194:405-416, 1979 44. Fregly MJ: Thyroid activity of spontaneous hypertensive rats. Proc Soc Exp Biol Med 149:124-132, 1975 45. Wright GL, Knecht E, Badger D, Samueloff S, Toraason M, Dukes-Dobas F: Oxygen consumption in the spontaneously hypertensive rat. Proc Soc Exp Biol Med 159:449-452, 1978 46. Marek J, Thoenes W, Motlik K: Lipoide Transformation der Mitochondrien in Nebennierenrindenzellen nach Aminoglutathimid (Elipten Ciba). Virchows Archiv [Cell Pathol] 6:116-131, 1970 47. Malamed S: Ultrastructure of the mammalian adrenal cortex in relation to secretory function, Handbook of Physiology. Section 7, Vol 6, Adrenal Gland. Edited by RO Greep, EB Astwood. American Physiological Society, Washington, DC, 1975, pp 25-39
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48. Sugihara H, Kawai K, Tsuchiyama H: Pathology of intracortical nodules in rat adrenal glands, especially on their fine-structure. Acta Pathol Jpn 23:253-260, 1973 49. Dobbie JW: Adrenocortical nodular hyperplasia: The ageing adrenal. J Pathol 99:1-18, 1969
Acknowledgments Mrs. Neonila Fylypiw, Mrs. Priscilla Adams, Mrs. Elizabeth Lawson, Mrs. Geneva Joseph, Mrs. Luise Bohacek, and Mr. Robert Linsmair provided skilled technical assistance. Mrs. Berta Cole typed the manuscript.
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[Illustrations follow]
Figure 1-Zona reticularis cell from an SH rat 70 weeks of age. sinusoids are filled with red blood cells (R). Parenchymal cells contain lipid droplets inProminent the cytoplasm readily identified by their electron-lucid matrix. (x5520) Figure 2-Zona reticularis cell from a WKY rat 70 weeks of age. A large single lipid droplet (L) is incorporated into a Mitochondrial cristae (C) are observed at one point; mitochondrial members aremitochondrion. double and attenuated, and contain no cristae at another point (arrows). Several accumulations of lipofuscin (F) are observed in the cytoplasm. Smooth endoplasmic reticulum (ER). (x 18,400) Inset-4n a 0.5-u toluidine-blue-stained section, some cells contain enlarged, clear vacuolar lipid droplets. (x725) (Both with a photographic reduction of 5%)
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of age. A Figure 3-Cell from a nodule within the zona reticularis of a WKY rat at 70 weeks limiting membrane, alsingle lipid droplet (L) occupies almost the entire cytoplasm. There is no of though discontinuous cisternae of smooth endoplasmic reticulum are seen at the periphery Figure 4-Cell from a the droplet (arrows). Mitochondrial cristae are lamellar. (xl 1,600) nodule within the zona reticularis of a WKY rat at 70 weeks of age. Mitochondria in some parenchymal cells are attenuated and contain lamellar cristae. (x1 2,600) (Both with a photographic reduction of 5%)
Figure 5-Zona reticularis from a WKY rat 95 weeks of age. Smooth endoplasmic reticulum (ER) is hypertrophic. Lipofuscin (F). (xi 6,000) Inset-An especially large A septum (arrow) isolates a peripheral area of the mitochrondrion. (X10,400) mitochondrion. Figure 6Zona reticularis cell from a WKY rat 95 weeks of age. A single large lipid droplet is encircled a mitochondrion. At one point, there is apparently a septation (arrow) of the mitochondria by surrounding the lipid droplet. (x 16,000) (Both with a photographic reduction of 5%)
Figure 7-Zona reticularis from a SH rat at 95 weeks of age. Hypertrophic smooth endoplasmic reticulum (ER) is arranged in concentric lamellar formations. Mitochondria contain small numFigure 8-Macrophage in the inner zona reticubers of tubulovesicular cristae. (x 17,600) laris adjacent to the medulla of a SH rat 95 weeks of age. The macrophage can be identified by small mitochondria with lamellar cristae. Discrete areas of lipofuscin (F) and membrane-bound vacuoles (V) with a moderately electron-opaque matrix can be seen throughout the cytoplasm. Membrane-bound granules with an electron-opaque core are present in a medullary cell (M). (x 17,600) (Both with a photographic reduction of 5%)
