The Adrenal Cortex in Spontaneously Hypertensive Rats A Quantitative U'ltrastructural Sttudy Peter A. Nickerson, MD
The adrenal cortex of spontaneously hypertensive rats (SHR) has been examined by quantitative morphologic techniques for electron microscopy. The volume and surface area of smooth endoplasmic reticulum and the volume of Golgi apparatuses in zona glomerulosa cells of SHR was significantly greater than those of Wistar-Kyoto strain (W/KY) normotensive controls; the volume of lipid droplets and nucleus was significantly less in SHR than in W/KY animals. A stimulation of the zona glomerulosa in SHR may well be attributable to the elevation in systolic blood pressure. A distinct lipid-free subglomerulosa was observed in the adrenal gland of WV/KY rats; the cell volume was similar to that of the zona glomerulosa although the cells showed a significantly greater volume of mitochondria and surface area of mitochondrial membranes and greater volume of smooth endoplasmic reticulum and lvsosomes. In the zona fasciculata, cell volume, volumes and surface area of mitochondria and smooth endoplasmic reticulum, and volume of lipid droplets were significantly lower in SHR than in W/KY rats. The volume of the Golgi apparatus was greater in SHR than in W/KY rats. Glycogen particles were observed in focal areas of some zona fasciculata cells. The adrenal cortex of another strain of normotensive Wistar rat (W/CFN) was compared with that of the W/KY and SHR. Although the relative adrenal weights of SHR and W/KY animals were identical, the weight of that in W/CFN was significantly smaller. The volume of the zona glomerulosa of SHR was significantly greater than that of W/KY although the volume of the zona glomerulosa in W/CFN was significantly greater than the other two groups. The volume of nucleus and lipid droplets of zona glomerulosa in W/KY was significantly greater than that in the W/CFN-; the volume of the cell, mitochondria, smooth endoplasmic reticulum, lipid droplets, and lysosomes, and the surface area of smooth endoplasmic reticulum and mitochondrial membranes of W/KY animals was significantly greater than those of W/CFN animals. It is concluded that the W/CFN rat is not an appropriate control for spontaneously hypertensive rats. (Am J Pathol 84:545-560, 1976)
OKAMIOTO XND AOKI 1 developed a strain of spontaneouslx- hypertensive rats (SHR) by selective breeding in a colony of WNistar animals. The SHR is an excellent model of human essential hypertension because no drug treatment or surgical procedure is required to raise the blood pressure. Pathologic changes in SHR include vascular disease, cardiomegalx-, nephrosclerosis, and cerebral hemorrhage.2 The endocrine glands may play- some role in the pathogenesis of the From the Department of Pathology. State U~niversity of Nev' -Work at Buffalo. Buffalo. Ne" 'York Supported by Grant HL-06975 from the National Heart and Lung Institute Xccepted for publication -\la\ 12. 1976 Xddress reprint requests to Dr Peter A Nickerson. Department of Patholog!. State U niversit% of New- 'tork at Buffalo. 10 Race Street. Buffalo. NY 14207
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hypertension. Fregly! ' has reported hypofunction of the thyroid gland in hypertensive SHR in spite of increased TSH secretion. Abnormalities in the pituitary-adrenal axis 4 and in steroid biosynthesis have been reported in SHR. In fact, differences between the adrenal gland of SHR and that of controls are already evident before birth.9 Aoki 6 observed that the adrenal gland is essential for the development of hypertension in SHR. On the other hand, Baer, Knowlton, and Laragh 10 reported that hypertension developed in spite of adrenalectomy and questioned whether the adrenal gland is essential for the development and maintenance of the hypertension in SHR. Maruvama " and Tsuchivama, Sugihara, and Kawai 12 observed a prominent smooth endoplasmic reticulum in zona fasciculata cells of SHR. NMoreover, an enlarged Golgi apparatus was reported in SHR.'2 Quantitative ultrastructural techniques provide an objective method for assessing changes in ultrastructure.13 Inasmuch as no quantitative studies of the adrenal cortex in SHR have appeared, it was the purpose of the present study to compare the ultrastructure of the adrenal gland in spontaneously hypertensive rats with that of two strains of normotensive Wistar rats. Materials and Methods Fifteen male SHR (CFSHR) and 15 male Wistar rats (XV CF'N) were obtained from Charles River Breeding Co., Wilmington, Mass. Fifteen normotensive male rats of the Wistar-Kyoto strain (W' KY) were purchased from Laboratory Supplx- Co. Inc.. Indianapolis, Ind. All animals were caged individually and given Purina Lab Chow and tap water ad libitum. Blood pressure was recorded in unanesthetized rats.14 Animals were sacrificed by decapitation at 21 weeks of age. The left adrenal gland from 6 W' CFN, 6 X' KY controls, and 6 SHR was removed rapidly and trimmed of adherent fat. The adrenal gland was cut into 1-mm slices and fixed in 3%c purified glutaraldehyde Ladd Research Industries, Burlington, Vt.) buffered to pH 7.4 with 0.1 NM phosphate. Pieces of zona glomerulosa and zona fasciculata were obtained with a microscalpel with aid of the three times magnification of an American Optical dissecting microscope 15 and processed for electron microscopy." Half-micron sections were cut from all blocks to verify the zonal position of the tissue. Thin sections w-ere cut with glass knives on a Porter-Blum MT-i ultramicrotome and stained with with uranyl acetate 16 and lead citrate 17 before examination with a Siemens 101 electron microscope. The methods of WJeibel and Bolender 1a were employed for quantitative analysis of electron micrographs. A multipurpose test pattem having 50 lines and 100 points was used to obtain fractional volumes and surface density. Six micrographs wvere taken each at 2400 x and 8000 x from five blocks for each animal and enlarged photographically to 8400 x and 28,000 X. The volume fraction of cytoplasm and nucleus w-as determined on the 8400 X prints, whereas the volume fractions of mitochondria, smooth endoplasmic reticulum, lipid droplets, Golgi apparatuses, and lsosomes w ere determined on the 28,000 X micrographs. The surface area of smooth endoplasmic reticulum and mitochondrial membranes was calculated by counting intersections of these structures with the test lines at 28,000 X.
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AXerage volume of zona glomerulosa and zona fasciculata cells 'sas determined b- the method of Nussdorfer 1 on photographs of 0.5- toluidine blue-stained sections. \olume of the zona glomerulosa vsas c-alculated bv the method of Nussdorfer. \lazzocchi and Rebuffat 1' on .5j sections stained X ith hematoxvlin and eosin. Measurements of the mean vsidth of the zona glomerulosa msere made on a central section cut at right angles to the long axis of the gland. Four measurements of the zona glomerulosa wsidth were made at 900 from each other at 450 X wsith an American Optical filiar micrometer. The diameter of the t\s-o axes of the section wsas also measured wsith the micrometer at 40 x. Zona glomerulosa volume wsas determined in six adrenal glands each from SHR and from b1oth control groups. Organs wsere fixed in 10c phosphate-buffered formalinl and wseighed after fixation. All data vsere expressed as the mean ± SE and analyzed statistically by the Student t test. Intragroup means of volumes and surface areas for cellular organefles were assessed bh the F ratio analysis of v ariance.
Results The body weight of rats in the SHR group did not differ from that of rats in the XW KY group but was significantly less than that of W CFN controls at the beginning of the experiment (Table 1!. Bx- 21 weeks. the bodx- weight of both control groups was significantl\- greater than that of SHR although the increase in body Xweight of XV CF'N animals w as especially pronounced. Systolic blood pressures of both control groups did not differ from those of SHR at the start of the experiment: the SHR group became hypertensive by 21 weeks of age. whereas the blood pressures of control animals did not differ from one another (Table 1) The wseight of the adrenal gland in V CFN- (44.5 ± 0.7 mg) did not differ significantly from that of SHR (44. ± 2.6 mg although adrenal w-eight in the W KY group (5:3.4 ± 1.2 mg) was significantly greater than that of the other tw o groups (P < 0.001 ). However, the relative adrenal weight of SHR (17.2 ± 1.1 mg 100 g body weight) wvas significantly greater than that of X CFN- controls (9.3 ± 0.6 mg 100 g body weight). The average w idth of the zona glomerulosa in XV CFN- controls (:3.4 ± 1.1 ) wvas significantly greater than that in SHR (44.3 ±0.9 p) P < 0.001. Table 1-Comparison of Body Weight, Blood Pressure, and Relative Organ Weights of Two Wistar Rat Control Groups With Spontaneously Hypertensive Rats Relative weights Systolic blood pressure (mm Hg) (mg/100 g body weight) Body weight (g) Initial Groups Final Initial Final Heart Kidney 147=2 467 9 83=3 113=2 307=20 644=13 Control (W/CFN) 100 =5 344 =8 80 =3 105 =4 364 = 10 763 = 17 Control(W/KY) p < 0.001 p < 0.001 p < 0.01 W/CFN vs. W/KY P < 0.001 NS NS 97 =2 SHR 172 =6 286=6 75 =2 486=15 888 =29 p < p P p < < 0.001 < < 0.001 W/CFN vs. SHR P 0.001 0.001 NS 0.001 P < 0.001 P < 0.001 P < 0.001 P < 0.001 W/KYvs. SHR NS NS NS. not significant; values are mean = SE.
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whereas the zona glomerulosa width in W/KY rats (34.1 ± 0.3 A) was significantly less (P < 0.001) than that of the other two groups. The volume of the zona glomerulosa was also determined inasmuch as Nussdorfer et ail.9 suggested that the width of the zone may not reflect accurately the size of the zone. The volume of the zona glomerulosa in W/CFN animals (1.21 ± 0.05 cu mm) was significantly greater than that in SHR (1.02 ± 0.06 cu mm) (P < 0.001); the volume of the zona glomerulosa in W/KY rats was significantly smaller than that of either of the two other groups (0.77 ± 0.02 cu mm) (P < 0.001). The relative weights of heart and kidneys in SHR were significantly greater than those of Wistar controls (Table 1). Ulbastucture d Zona Gl1nulosa
It is of interest that the volume of the nucleus and lipid droplets in zona glomerulosa cells of W/KY rats was significantly greater than that of W/CFN animals, whereas the volume of mitochondria was significantly lower in W/KY than in W/CFN rats (Table 2; Figure 1). The surface area of smooth endoplasmic reticulum and mitochondrial membranes of the two control groups did not differ from one another (Table 3). The subglomerulosa zone was particularly promiment in W/KY control animals (Figure 2) because of an almost complete absence of lipid droplets; the subglomerulosa cells were not readily identifiable in the other two groups. The volume of mitochondria, smooth endoplasmic reticulum, lysosomes, and Golgi apparatus of W/KY subglomerulosa cells was significantly greater than that in adjacent zona glomerulosa cells. Similarly, the surface area of smooth endoplasmic reticulum and mitochondrial membranes in subglomerulosa cells was greater than that in the zona glomerulosa. The average volume of zona glomerulosa cells in SHR was significantly smaller than that in W/KY controls, although there was no significant difference in comparison to W/CFN rats (Table 2). The volume of smooth endoplasmic reticulum and Golgi apparatus in SHR was greater than that in W/KY rats, whereas the volumes of nucleus and lipid droplets were significantly less in SHR as compared to those in W/KY rats (Table 2; Figure 3). The W/CFN control, however, showed significantly smaller volumes of smooth endoplasmic reticulum and greater volumes of Golgi apparatus and lysosomes, but no significant alteration in average cellular volume or lipid droplets. A significantly greater surface area of smooth endoplasmic reticulum was observed in the SHR group although no significant changes occurred in mitochondrial membranes (Table 3).
ADRENAL CORTEX IN SHR
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Table 3-Surface Area of Cellular Organelles (sq w/cell) of Zona Glomerulosa, Sub-Zona Glomerulosa, and Zona Fasciculata SER Group Mitochondrial membranes* Zona glomerulosa Control (W/CFN) 1,868 i 305 5,042 ± 609 Control(W/KY) 1,912 109 4,211 i 326 W/CFN vs. W/KY NS NS SHR 3,168 ± 401 3,895 ± 648 W/CFN vs. SHR P < 0.05 NS p < 0.01 W/KY vs. SHR NS Sub-zona glormerulosa Control (W/KY) 3,006 ± 184 6,290 z 439 Zona fasciculata Control (W/CFN) 20,838 ± 700 9.487 ± 356 Control (W/KY) 13,828 ± 420 34,944 ± 1638 W/CFN vs. W/KY P < 0.001 P < 0.001 SHR 11,310 ± 440 21,679 ±911 P < 0.001 W/CFN vs. SHR NS p < 0.001 W/KY vs. SHR P < 0.001 NS, not significant; values are mean ± SE. Inner and outer membranes plus cristae.
Ultrastructure of Zona Fasciculata
The volume of the cell, nucleus, mitochondria. smooth endoplasmic reticulum, lipid droplets, and lysosomes of zona fasciculata cells in XN' KY animals (Figure 4) was significantly greater than that in WV CFN animals (Table 2). The surface area of smooth endoplasmic reticulum and mitochondrial membranes in XV' KY was significantly greater than that of XV CFN (Table 3). The volumes of the cell, mitochondria, smooth endoplasmic reticulum. l-sosomes, and Golgi apparatus of SHR were significantly greater than those of V /CFN controls; how ever, the volumes of the cell, mitochondria, and lipid droplets of SHR were significantly less than those of XN' KY rats (Figure 3); the volume of the Golgi apparatus of SHR was significantly greater than that of XV/KY rats (Table 2). The surface area of smooth endoplasmic reticulum of SHR %vas significantly greater than that in XV CFN animals but significantly less than that in XV /KY controls (Table 3). The surface area of mitochondrial membranes in SHR wvas significantly less than that in XV' KY animals w-hereas there X as no difference as compared to XV CFN controls. Increases in glvcogen particles were observed in focal areas of some zona fasciculata cells (Figure 6). Discussion
The XVistar strain obtained from Charles River Breeding Co. (X' CFN) is not an appropriate control for the SHR group. Volume of zona fasciculata cells, mitochondria, smooth endoplasmic reticulum, and lysosomes of
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W/KY rats differed considerably from those of W/CFN animals. Similarly, surface area of smooth endoplasmic reticulum and mitochondrial membranes of W/KY differed considerably from those of W/CFN. Freeman et al.8 have indicated that ordinary Wistar rats may differ with regard to response to stress and adrenal sensitivitv from W/KY animals. Similarly, Goldenberg "O reported that the incidence of gastric lesions was significantly higher in the SHR than in normotensive Wistar rats. The W/KY normotensive rats are derived from the original colony used to breed the SHR 1 and are in all probability closer in genetic constitution to the SHR than are other strains of Wistar animals. In our study, the control W/KY were obtained from Laboratory Supply Co., whereas the W/CFN and SHR were purchased from Charles River Breeding Co. because W/KY rats were not readilv available. In spite of different sources, rats were treated in the same manner insofar as possible and were sacrificed at identical ages. The volume of the zona fasciculata cells of W/KY rats was significantly greater than that of SHR. Surface area of smooth endoplasmic reticulum of SHR decreased significantly in comparison to that of W/KY controls. This finding contrasts with the report of well-developed smooth endoplasmic reticulum in SHR by Tsuchiyama et al.'2 Nonetheless, it should be mentioned that stereologic techniques offer an objective means of assessing changes in ultrastructure.13 Furthermore, quantitative studies require extensive random sampling of the tissue. The smooth endoplasmic reticulum contains enzvmes involved in a number of reactions in steroid synthesis. Several reactions in the svnthesis of cholesterol 21 and the conversion of pregnenolone to progesterone 2 and subsequently to deoxycorticosterone23 occur in the smooth reticulum. In spite of the decreased surface area of smooth endoplasmic reticulum, no change in secretion rate of corticosterone or deoxycorticosterone was observed by Freeman et al.' at 22 to 25 weeks of age. It should be noted that Moll et al.7 observed a suppression of 18-OH-i 1-deoxvcorticosterone and corticosterone secretion rates in SHR, and that Rapp and Dahl6 showed a decreased secretion rate of deoxycorticosterone. However, control animals were a Wistar strain of rats, not the W/KY animals. Similarly, a significantly smaller volume and surface area of mitochondria was observed in SHR. A decreased number of mitochondrial cristae has been observed in our laboratorv in adrenal regeneration hvpertension,'5 methylandrostenediol-induced hypertension,2' and in hvpertension in animals bearing a tumor secreting ACTH, growth hormone, and prolactin." However, abnormalities in adrenocortical function accompany the alterations in ultrastructure in all of these models.2" Nonetheless, no
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significant changes in corticosteroidogenesis have been observed in SHR as compared to W/KY controls at the time period we have studied. The volume of lipid droplets was decreased signficantly in SHR but increased appreciably in W/KY controls. The lipid droplets in the adrenal gland contain cholesterol and cholesterol esters and provide precursors for
steroidogenesis.X The volume of the Golgi apparatus was increased in SHR. Tsuchiyama et al.u have indicated that the Golgi apparatus is prominent in SHR. The function of the Golgi apparatus in steroidogenesis is not completely clear, although Long and Jones" have suggested that it plays a role in the sulfation of steroids. The width of the zona glomerulosa was significantly greater in SHR than that in W/KY rats despite a significantly smaller average volume of zona glomerulosa cells in SHR. Aoki et al.4 also observed that the crosssectional area of the zona glomerulosa in a central section of adrenal gland in SHR at 4 to 5 months of age was significantly larger than that of controls, although volume of the zone was not calculated. It is of particular interest that the width and volume of the zona glomerulosa have been directly related to the function of the zone; increased width is related to increased functional activity of the zone.2"-0 However, Freeman, et al." observed no significant change in aldosterone secretion rate at 22 to 25 weeks of age, although a significant decrease was observed in SHR at 7 to 8 and 11 to 13 weeks of age. The greater volume and surface area in the zona glomerulosa of SHR supports a stimulation of these cells. Smooth endoplasmic reticulum in zona glomerulosa cells hypertrophies in response to stimulation of the cells by sodium depletion;30.31 concomitantlv, there is an increased secretion of aldosterone.2' Nonetheless, the alterations in zona glomerulosa cells of SHR are relatively mild compared to those observed after sodium depletion. The zona glomerulosa of SHR may well show stimulation as a response to the elevation in blood pressure. Partial support for this hypothesis is provided by the recent report of Kasemsri and Nickerson n that the width of the zona glomerulosa in the adrenal cortex of rats made hypertensive by bilateral encapsulation of the kidneys is directly correlated with the height of the pressure attained. Similar increases in smooth endoplasmic reticulum also occur in renal encapsulation-induced hypertension.n Baer et al.10 have also suggested that secondary alterations occur in the adrenal gland of SRH as a result of the hypertension. It is unlikely that the renin-angiotensin system causes the changes in the zona glomerulosa inasmuch as Koletsky et al."3 and Freeman et al." showed significant decreases in plasma renin activity.
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No significant increase in adrenal weight was observed in SHR. The relative weight of the adrenal gland in SHR was signficantly greater than that of W/CFN rats but did not differ from that of W/KY rats. The reason for this difference is principally attributable to the relative weight of the W/KY adrenal gland. In the report of Aoki et al.,4 the relative adrenal weight was reported as 17 mg/100 g of body weight, which is identical to that observed in our experiment. However, the weight of the gland in Aoki's control was 13.9 mg/100 g of body weight,4 whereas in the present experiment it was 15.5 mg/100 g of body weight. The subglomerulosa of W/KY animals was particularly prominent because of the virtual absence of lipid droplets. The function of this region is obscure, although mitosis occurs frequently in this region.' Our studies demonstrate that the cells have some characteristics of both neighboring zones: they have the same volume as those of the zona glomerulosa but increased volume of mitochondria and smooth endoplasmic reticulum, similar to those of the zona fasciculata. The cells in the subglomerulosa region may well represent transitional cells. Support for a transformation of zona glomerulosa cells into those of the zona fasciculata comes from the studies of Kahri." Newborn adrenocortical cells maintained in tissue culture have characteristics of the zona glomerulosa but become transformed into those with typical characteristics of the zona fasciculata upon addition of ACTH.
Refereles 1. Okamoto K, Aoki K: Development of a strain of spontaneously hypertensive rats. Jap Circ J 27:282-293, 1963 2. Okamoto K, Aoki K, Nosaka S, Fukushima NI: Cardiovascular diseases in the spontaneously hypertensive rat. Jap Circ J 28:943-952, 1964 3. Fregly MJ: Thyroid activity of spontaneous hy pertensive rats. Proc Soc Exp Biol Nied 149:124-132, 1975 4. Aoki K, Tankawa H, Fujinami T, Miyazaki A, Hashimoto Y: Pathological studies on the endocrine organs of the spontaneously hypertensive rats. Jap Heart J 4:426-442, 1963 5. Aoki K: Experimental studies on the relationship between endocrine organs and hypertension in spontaneously hypertensive rats. I. Effect of hypophvsectomv, adrenalectomv, thvroidectomv, nephrectomy and sympathectomy on blood pressure. Jap Heart J 4:443461, 1963 6. Rapp JP, Dahl LK: Adrenal steroidogenesis in rats bred for susceptibility and resistance to the hypertensive effect of salt. Endocrinology 88:32-65, 1971 7. NMoll 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, U lick S, Weinberger NIH: Control of aldosterone secretion in the spontaneously hypertensive rat. Circ Res 37:66-71, 1975 9. Taira Y: Histological and histoautoradiographical studies on the fetal adrenal cortex in spontaneously hypertensive rats. Acta Pathol Jap 24:733-746, 1974
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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, p 203 11. Maruyama T: Electron microscopic studies on the adrenal medulla and adrenal cortex of hypertensive rats. I. Spontaneously hypertensive rats. jap Circ J 33:1271-1284, 1969 12. Tsuchiyama H, Sugihara H, Kawai K: Pathology of the adrenal cortex in spontaneously hypertensive rats."" p 177 13. 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 Company, 1973, pp 237-296 14. Molteni A, Brownie AC, Skelton FR: Production of hypertensive vascular disease in the rat by methyltestosterone. Lab Invest 21:129-137, 1969 15. Nickerson PA, Brownie AC, Skelton FR: An electron microscopic studv of the regenerating adrenal gland during the development of adrenal regeneration hypertension. Am J Pathol 57:335-364, 1969 16. Stempak JG, Ward RT: An improved staining method for electron microscopy. J Cell Biol 22:697-701, 1964 17. 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 18. Nussdorfer GG: Effects of corticosteroid-hornones on the smooth endoplasmic reticulum of rat adrenocortical cells. Z Zellforsch Mikrosk Anat 106:143-154, 1970 19. Nussdorfer GG, Mazzocchi G, Rebuffat P: An ultrastructural stereologic study of the effects of ACTH and adenosine 3',5'-cyclic monophosphate on the zona glomerulosa of rat adrenal cortex. Endocrinology 92:141-151, 1973 20. Goldenberg MM: Study of cold plus restraint stress gastric lesions in spontaneouslv hypertensive, Wistar and Sprague-Dawley rats. Life Sci 12:519-527, 1973 21. 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 22. Bever KF, Samuels LT: Distribution of steroid-,B-ol-dehydrogenase in cellular structures of the adrenal gland. J Biol Chem 219:69-76, 1956 23. Ryan KJ, Engel LL: Hydroxylation of steroids at carbon 21. J Biol Chem 225:103-114, 1957 24. Brownie AC, Skelton FR: Adrenocortical function and structure in adrenal-regeneration and methyladrostenediol hypertension. Functions of the Adrenal Cortex. Edited by KW McKems. New York, Appleton-Centurv-Crofts, 1968, p 691 25. 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 26. Moses HL, Davis WW, Rosenthal AS, Garren LD: Adrenal cholesterol: Localization by electron microscope autoradiography. Science 163:1203-1206, 1969 27. Long JA, Jones AL: The fine structure of the zona glomerulosa and the zona fasciculata of the adrenal cortex of the opossum. Am J Anat 120:463-488, 1967 28. Deane HW, Shaw JH, Greep RO: The effect of altered sodium or potassium intake on the width and cytochemistry of the zona glomerulosa of the rat's adrenal cortex, Endocrinology 43:133-153, 1948 29. Hartroft PM, Eisenstein AB: Alterations in the adrenal cortex of the rat induced bysodium deficiency: Correlation of histologic changes with steroid hormone secretion. Endocrinology 60:641-651, 1957 30. Giacomelli F, Wiener J, Spiro D: Cytological alterations related to stimulation of the zona glomerulosa of the adrenal gland. J Cell Biol 26:499-522, 1965
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31. Long JA, Jones AL: Alterations in fine structure of the opossum adrenal cortex following sodium deprivation. Anat Rec 166:1-26, 1970 32. Kasemsri S, Nickerson PA: Quantitative ultrastructural studv of the rat adrenal cortex in renal encapsulation-induced hypertension. Am J Pathol 82:143-156, 1976 33. Koletskv S, Shook P, Rivera-Velez J: Lack of increased renin-angiotensin activitv in rats with spontaneous hypertension. Proc Soc Exp Biol Med 134:1187-1190, 1970 34. Brenner RM: Radioautographic studies with tritiated thymidine of cell migration in the mouse adrenal after a carbon tetrachloride stress. Am J Anat 112:81-95, 1963 35. Kahri A: Histochemical and electron microscopic studies on the cells of the rat adrenal cortex in tissue culture. Acta Endocrinol [Suppl 108] (kbh) 52:3-96, 1966
Acknowledgments The author is grateful to Neonile Fylypiw, Luther Joseph, Geneva Joseph, Elisabeth Lawson, and Robert Linsmair for skilled technical assistance. Berta Cole assisted with typing of the manuscript.
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[Illusatrao follow]
f-~
,
-
Figure 1-Zona glomerulosa cells from W/KY rat. Numerous lipid droplets (L) are observed in all of the zona glomerulosa cells. Mitochondria are circular to elongate in contour. Endoplasmic reticulum is predominantly smooth surfaced (ER). L Y = lysomsome, G = Golgi apparatus. ( x 16,000) Figure 2Cells in the subglomerulosa of W/KY animal. The cells are lipid free. Numerous cisternae of smooth endoplasmic reticulum (ER) are dispersed throughout the cell. Lysosomes (arrow) are numerous. G = Golgi apparatus, S = sinusoid. (x 16,000)
Figure 3-Zona glomerulosa cells from SHR. Many lipid droplets (L) are observed within the cells. Smooth endoplasmic reticulum (ER). One mitochondrion contains cristae which appear as straight tubules in section (arrow). (x 22,400)
Figure 4-Zona fasciculata cell from W/KY rat. Lipid droplets (L) are particularly numerous. Mitochondria are circular in contour and contain vesicular cristae. ER reticulum. (x 16,000)
=
smooth endoplasmic
3
4
5
Figure 5-Zona fasciculata cell from SHR. Smooth endoplasmic reticulum (ER) is scattered throughout Figure 6the cell. Mitochondrial cristae are tubulovesicular (arrow). L = lipid droplets. (x 22,400) The Zona fasciculata cells from SHR. Focal areas of glycogen (GL) are observed in the cytoplasm. Golgi apparatus (G) is particularly prominent. ER = smooth endoplasmic reticulum LY = lysosomes. (x 20,800)
The adrenal cortex of spontaneously hypertensive rats (SHR) has been examined by quantitative morphologic techniques for electron microscopy. The volume and surface area of smooth endoplasmic reticulum and the volume of Golgi apparatuses in zona glomerulosa cells of SHR was significantly greater than those of Wistar-Kyoto strain (W/KY) normotensive controls; the volume of lipid droplets and nucleus was significantly less in SHR than in W/KY animals. A stimulation of the zona glomerulosa in SHR may well be attributable to the elevation in systolic blood pressure. A distinct lipid-free subglomerulosa was observed in the adrenal gland of WV/KY rats; the cell volume was similar to that of the zona glomerulosa although the cells showed a significantly greater volume of mitochondria and surface area of mitochondrial membranes and greater volume of smooth endoplasmic reticulum and lvsosomes. In the zona fasciculata, cell volume, volumes and surface area of mitochondria and smooth endoplasmic reticulum, and volume of lipid droplets were significantly lower in SHR than in W/KY rats. The volume of the Golgi apparatus was greater in SHR than in W/KY rats. Glycogen particles were observed in focal areas of some zona fasciculata cells. The adrenal cortex of another strain of normotensive Wistar rat (W/CFN) was compared with that of the W/KY and SHR. Although the relative adrenal weights of SHR and W/KY animals were identical, the weight of that in W/CFN was significantly smaller. The volume of the zona glomerulosa of SHR was significantly greater than that of W/KY although the volume of the zona glomerulosa in W/CFN was significantly greater than the other two groups. The volume of nucleus and lipid droplets of zona glomerulosa in W/KY was significantly greater than that in the W/CFN-; the volume of the cell, mitochondria, smooth endoplasmic reticulum, lipid droplets, and lysosomes, and the surface area of smooth endoplasmic reticulum and mitochondrial membranes of W/KY animals was significantly greater than those of W/CFN animals. It is concluded that the W/CFN rat is not an appropriate control for spontaneously hypertensive rats. (Am J Pathol 84:545-560, 1976)
OKAMIOTO XND AOKI 1 developed a strain of spontaneouslx- hypertensive rats (SHR) by selective breeding in a colony of WNistar animals. The SHR is an excellent model of human essential hypertension because no drug treatment or surgical procedure is required to raise the blood pressure. Pathologic changes in SHR include vascular disease, cardiomegalx-, nephrosclerosis, and cerebral hemorrhage.2 The endocrine glands may play- some role in the pathogenesis of the From the Department of Pathology. State U~niversity of Nev' -Work at Buffalo. Buffalo. Ne" 'York Supported by Grant HL-06975 from the National Heart and Lung Institute Xccepted for publication -\la\ 12. 1976 Xddress reprint requests to Dr Peter A Nickerson. Department of Patholog!. State U niversit% of New- 'tork at Buffalo. 10 Race Street. Buffalo. NY 14207
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hypertension. Fregly! ' has reported hypofunction of the thyroid gland in hypertensive SHR in spite of increased TSH secretion. Abnormalities in the pituitary-adrenal axis 4 and in steroid biosynthesis have been reported in SHR. In fact, differences between the adrenal gland of SHR and that of controls are already evident before birth.9 Aoki 6 observed that the adrenal gland is essential for the development of hypertension in SHR. On the other hand, Baer, Knowlton, and Laragh 10 reported that hypertension developed in spite of adrenalectomy and questioned whether the adrenal gland is essential for the development and maintenance of the hypertension in SHR. Maruvama " and Tsuchivama, Sugihara, and Kawai 12 observed a prominent smooth endoplasmic reticulum in zona fasciculata cells of SHR. NMoreover, an enlarged Golgi apparatus was reported in SHR.'2 Quantitative ultrastructural techniques provide an objective method for assessing changes in ultrastructure.13 Inasmuch as no quantitative studies of the adrenal cortex in SHR have appeared, it was the purpose of the present study to compare the ultrastructure of the adrenal gland in spontaneously hypertensive rats with that of two strains of normotensive Wistar rats. Materials and Methods Fifteen male SHR (CFSHR) and 15 male Wistar rats (XV CF'N) were obtained from Charles River Breeding Co., Wilmington, Mass. Fifteen normotensive male rats of the Wistar-Kyoto strain (W' KY) were purchased from Laboratory Supplx- Co. Inc.. Indianapolis, Ind. All animals were caged individually and given Purina Lab Chow and tap water ad libitum. Blood pressure was recorded in unanesthetized rats.14 Animals were sacrificed by decapitation at 21 weeks of age. The left adrenal gland from 6 W' CFN, 6 X' KY controls, and 6 SHR was removed rapidly and trimmed of adherent fat. The adrenal gland was cut into 1-mm slices and fixed in 3%c purified glutaraldehyde Ladd Research Industries, Burlington, Vt.) buffered to pH 7.4 with 0.1 NM phosphate. Pieces of zona glomerulosa and zona fasciculata were obtained with a microscalpel with aid of the three times magnification of an American Optical dissecting microscope 15 and processed for electron microscopy." Half-micron sections were cut from all blocks to verify the zonal position of the tissue. Thin sections w-ere cut with glass knives on a Porter-Blum MT-i ultramicrotome and stained with with uranyl acetate 16 and lead citrate 17 before examination with a Siemens 101 electron microscope. The methods of WJeibel and Bolender 1a were employed for quantitative analysis of electron micrographs. A multipurpose test pattem having 50 lines and 100 points was used to obtain fractional volumes and surface density. Six micrographs wvere taken each at 2400 x and 8000 x from five blocks for each animal and enlarged photographically to 8400 x and 28,000 X. The volume fraction of cytoplasm and nucleus w-as determined on the 8400 X prints, whereas the volume fractions of mitochondria, smooth endoplasmic reticulum, lipid droplets, Golgi apparatuses, and lsosomes w ere determined on the 28,000 X micrographs. The surface area of smooth endoplasmic reticulum and mitochondrial membranes was calculated by counting intersections of these structures with the test lines at 28,000 X.
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AXerage volume of zona glomerulosa and zona fasciculata cells 'sas determined b- the method of Nussdorfer 1 on photographs of 0.5- toluidine blue-stained sections. \olume of the zona glomerulosa vsas c-alculated bv the method of Nussdorfer. \lazzocchi and Rebuffat 1' on .5j sections stained X ith hematoxvlin and eosin. Measurements of the mean vsidth of the zona glomerulosa msere made on a central section cut at right angles to the long axis of the gland. Four measurements of the zona glomerulosa wsidth were made at 900 from each other at 450 X wsith an American Optical filiar micrometer. The diameter of the t\s-o axes of the section wsas also measured wsith the micrometer at 40 x. Zona glomerulosa volume wsas determined in six adrenal glands each from SHR and from b1oth control groups. Organs wsere fixed in 10c phosphate-buffered formalinl and wseighed after fixation. All data vsere expressed as the mean ± SE and analyzed statistically by the Student t test. Intragroup means of volumes and surface areas for cellular organefles were assessed bh the F ratio analysis of v ariance.
Results The body weight of rats in the SHR group did not differ from that of rats in the XW KY group but was significantly less than that of W CFN controls at the beginning of the experiment (Table 1!. Bx- 21 weeks. the bodx- weight of both control groups was significantl\- greater than that of SHR although the increase in body Xweight of XV CF'N animals w as especially pronounced. Systolic blood pressures of both control groups did not differ from those of SHR at the start of the experiment: the SHR group became hypertensive by 21 weeks of age. whereas the blood pressures of control animals did not differ from one another (Table 1) The wseight of the adrenal gland in V CFN- (44.5 ± 0.7 mg) did not differ significantly from that of SHR (44. ± 2.6 mg although adrenal w-eight in the W KY group (5:3.4 ± 1.2 mg) was significantly greater than that of the other tw o groups (P < 0.001 ). However, the relative adrenal weight of SHR (17.2 ± 1.1 mg 100 g body weight) wvas significantly greater than that of X CFN- controls (9.3 ± 0.6 mg 100 g body weight). The average w idth of the zona glomerulosa in XV CFN- controls (:3.4 ± 1.1 ) wvas significantly greater than that in SHR (44.3 ±0.9 p) P < 0.001. Table 1-Comparison of Body Weight, Blood Pressure, and Relative Organ Weights of Two Wistar Rat Control Groups With Spontaneously Hypertensive Rats Relative weights Systolic blood pressure (mm Hg) (mg/100 g body weight) Body weight (g) Initial Groups Final Initial Final Heart Kidney 147=2 467 9 83=3 113=2 307=20 644=13 Control (W/CFN) 100 =5 344 =8 80 =3 105 =4 364 = 10 763 = 17 Control(W/KY) p < 0.001 p < 0.001 p < 0.01 W/CFN vs. W/KY P < 0.001 NS NS 97 =2 SHR 172 =6 286=6 75 =2 486=15 888 =29 p < p P p < < 0.001 < < 0.001 W/CFN vs. SHR P 0.001 0.001 NS 0.001 P < 0.001 P < 0.001 P < 0.001 P < 0.001 W/KYvs. SHR NS NS NS. not significant; values are mean = SE.
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whereas the zona glomerulosa width in W/KY rats (34.1 ± 0.3 A) was significantly less (P < 0.001) than that of the other two groups. The volume of the zona glomerulosa was also determined inasmuch as Nussdorfer et ail.9 suggested that the width of the zone may not reflect accurately the size of the zone. The volume of the zona glomerulosa in W/CFN animals (1.21 ± 0.05 cu mm) was significantly greater than that in SHR (1.02 ± 0.06 cu mm) (P < 0.001); the volume of the zona glomerulosa in W/KY rats was significantly smaller than that of either of the two other groups (0.77 ± 0.02 cu mm) (P < 0.001). The relative weights of heart and kidneys in SHR were significantly greater than those of Wistar controls (Table 1). Ulbastucture d Zona Gl1nulosa
It is of interest that the volume of the nucleus and lipid droplets in zona glomerulosa cells of W/KY rats was significantly greater than that of W/CFN animals, whereas the volume of mitochondria was significantly lower in W/KY than in W/CFN rats (Table 2; Figure 1). The surface area of smooth endoplasmic reticulum and mitochondrial membranes of the two control groups did not differ from one another (Table 3). The subglomerulosa zone was particularly promiment in W/KY control animals (Figure 2) because of an almost complete absence of lipid droplets; the subglomerulosa cells were not readily identifiable in the other two groups. The volume of mitochondria, smooth endoplasmic reticulum, lysosomes, and Golgi apparatus of W/KY subglomerulosa cells was significantly greater than that in adjacent zona glomerulosa cells. Similarly, the surface area of smooth endoplasmic reticulum and mitochondrial membranes in subglomerulosa cells was greater than that in the zona glomerulosa. The average volume of zona glomerulosa cells in SHR was significantly smaller than that in W/KY controls, although there was no significant difference in comparison to W/CFN rats (Table 2). The volume of smooth endoplasmic reticulum and Golgi apparatus in SHR was greater than that in W/KY rats, whereas the volumes of nucleus and lipid droplets were significantly less in SHR as compared to those in W/KY rats (Table 2; Figure 3). The W/CFN control, however, showed significantly smaller volumes of smooth endoplasmic reticulum and greater volumes of Golgi apparatus and lysosomes, but no significant alteration in average cellular volume or lipid droplets. A significantly greater surface area of smooth endoplasmic reticulum was observed in the SHR group although no significant changes occurred in mitochondrial membranes (Table 3).
ADRENAL CORTEX IN SHR
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Table 3-Surface Area of Cellular Organelles (sq w/cell) of Zona Glomerulosa, Sub-Zona Glomerulosa, and Zona Fasciculata SER Group Mitochondrial membranes* Zona glomerulosa Control (W/CFN) 1,868 i 305 5,042 ± 609 Control(W/KY) 1,912 109 4,211 i 326 W/CFN vs. W/KY NS NS SHR 3,168 ± 401 3,895 ± 648 W/CFN vs. SHR P < 0.05 NS p < 0.01 W/KY vs. SHR NS Sub-zona glormerulosa Control (W/KY) 3,006 ± 184 6,290 z 439 Zona fasciculata Control (W/CFN) 20,838 ± 700 9.487 ± 356 Control (W/KY) 13,828 ± 420 34,944 ± 1638 W/CFN vs. W/KY P < 0.001 P < 0.001 SHR 11,310 ± 440 21,679 ±911 P < 0.001 W/CFN vs. SHR NS p < 0.001 W/KY vs. SHR P < 0.001 NS, not significant; values are mean ± SE. Inner and outer membranes plus cristae.
Ultrastructure of Zona Fasciculata
The volume of the cell, nucleus, mitochondria. smooth endoplasmic reticulum, lipid droplets, and lysosomes of zona fasciculata cells in XN' KY animals (Figure 4) was significantly greater than that in WV CFN animals (Table 2). The surface area of smooth endoplasmic reticulum and mitochondrial membranes in XV' KY was significantly greater than that of XV CFN (Table 3). The volumes of the cell, mitochondria, smooth endoplasmic reticulum. l-sosomes, and Golgi apparatus of SHR were significantly greater than those of V /CFN controls; how ever, the volumes of the cell, mitochondria, and lipid droplets of SHR were significantly less than those of XN' KY rats (Figure 3); the volume of the Golgi apparatus of SHR was significantly greater than that of XV/KY rats (Table 2). The surface area of smooth endoplasmic reticulum of SHR %vas significantly greater than that in XV CFN animals but significantly less than that in XV /KY controls (Table 3). The surface area of mitochondrial membranes in SHR wvas significantly less than that in XV' KY animals w-hereas there X as no difference as compared to XV CFN controls. Increases in glvcogen particles were observed in focal areas of some zona fasciculata cells (Figure 6). Discussion
The XVistar strain obtained from Charles River Breeding Co. (X' CFN) is not an appropriate control for the SHR group. Volume of zona fasciculata cells, mitochondria, smooth endoplasmic reticulum, and lysosomes of
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W/KY rats differed considerably from those of W/CFN animals. Similarly, surface area of smooth endoplasmic reticulum and mitochondrial membranes of W/KY differed considerably from those of W/CFN. Freeman et al.8 have indicated that ordinary Wistar rats may differ with regard to response to stress and adrenal sensitivitv from W/KY animals. Similarly, Goldenberg "O reported that the incidence of gastric lesions was significantly higher in the SHR than in normotensive Wistar rats. The W/KY normotensive rats are derived from the original colony used to breed the SHR 1 and are in all probability closer in genetic constitution to the SHR than are other strains of Wistar animals. In our study, the control W/KY were obtained from Laboratory Supply Co., whereas the W/CFN and SHR were purchased from Charles River Breeding Co. because W/KY rats were not readilv available. In spite of different sources, rats were treated in the same manner insofar as possible and were sacrificed at identical ages. The volume of the zona fasciculata cells of W/KY rats was significantly greater than that of SHR. Surface area of smooth endoplasmic reticulum of SHR decreased significantly in comparison to that of W/KY controls. This finding contrasts with the report of well-developed smooth endoplasmic reticulum in SHR by Tsuchiyama et al.'2 Nonetheless, it should be mentioned that stereologic techniques offer an objective means of assessing changes in ultrastructure.13 Furthermore, quantitative studies require extensive random sampling of the tissue. The smooth endoplasmic reticulum contains enzvmes involved in a number of reactions in steroid synthesis. Several reactions in the svnthesis of cholesterol 21 and the conversion of pregnenolone to progesterone 2 and subsequently to deoxycorticosterone23 occur in the smooth reticulum. In spite of the decreased surface area of smooth endoplasmic reticulum, no change in secretion rate of corticosterone or deoxycorticosterone was observed by Freeman et al.' at 22 to 25 weeks of age. It should be noted that Moll et al.7 observed a suppression of 18-OH-i 1-deoxvcorticosterone and corticosterone secretion rates in SHR, and that Rapp and Dahl6 showed a decreased secretion rate of deoxycorticosterone. However, control animals were a Wistar strain of rats, not the W/KY animals. Similarly, a significantly smaller volume and surface area of mitochondria was observed in SHR. A decreased number of mitochondrial cristae has been observed in our laboratorv in adrenal regeneration hvpertension,'5 methylandrostenediol-induced hypertension,2' and in hvpertension in animals bearing a tumor secreting ACTH, growth hormone, and prolactin." However, abnormalities in adrenocortical function accompany the alterations in ultrastructure in all of these models.2" Nonetheless, no
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significant changes in corticosteroidogenesis have been observed in SHR as compared to W/KY controls at the time period we have studied. The volume of lipid droplets was decreased signficantly in SHR but increased appreciably in W/KY controls. The lipid droplets in the adrenal gland contain cholesterol and cholesterol esters and provide precursors for
steroidogenesis.X The volume of the Golgi apparatus was increased in SHR. Tsuchiyama et al.u have indicated that the Golgi apparatus is prominent in SHR. The function of the Golgi apparatus in steroidogenesis is not completely clear, although Long and Jones" have suggested that it plays a role in the sulfation of steroids. The width of the zona glomerulosa was significantly greater in SHR than that in W/KY rats despite a significantly smaller average volume of zona glomerulosa cells in SHR. Aoki et al.4 also observed that the crosssectional area of the zona glomerulosa in a central section of adrenal gland in SHR at 4 to 5 months of age was significantly larger than that of controls, although volume of the zone was not calculated. It is of particular interest that the width and volume of the zona glomerulosa have been directly related to the function of the zone; increased width is related to increased functional activity of the zone.2"-0 However, Freeman, et al." observed no significant change in aldosterone secretion rate at 22 to 25 weeks of age, although a significant decrease was observed in SHR at 7 to 8 and 11 to 13 weeks of age. The greater volume and surface area in the zona glomerulosa of SHR supports a stimulation of these cells. Smooth endoplasmic reticulum in zona glomerulosa cells hypertrophies in response to stimulation of the cells by sodium depletion;30.31 concomitantlv, there is an increased secretion of aldosterone.2' Nonetheless, the alterations in zona glomerulosa cells of SHR are relatively mild compared to those observed after sodium depletion. The zona glomerulosa of SHR may well show stimulation as a response to the elevation in blood pressure. Partial support for this hypothesis is provided by the recent report of Kasemsri and Nickerson n that the width of the zona glomerulosa in the adrenal cortex of rats made hypertensive by bilateral encapsulation of the kidneys is directly correlated with the height of the pressure attained. Similar increases in smooth endoplasmic reticulum also occur in renal encapsulation-induced hypertension.n Baer et al.10 have also suggested that secondary alterations occur in the adrenal gland of SRH as a result of the hypertension. It is unlikely that the renin-angiotensin system causes the changes in the zona glomerulosa inasmuch as Koletsky et al."3 and Freeman et al." showed significant decreases in plasma renin activity.
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No significant increase in adrenal weight was observed in SHR. The relative weight of the adrenal gland in SHR was signficantly greater than that of W/CFN rats but did not differ from that of W/KY rats. The reason for this difference is principally attributable to the relative weight of the W/KY adrenal gland. In the report of Aoki et al.,4 the relative adrenal weight was reported as 17 mg/100 g of body weight, which is identical to that observed in our experiment. However, the weight of the gland in Aoki's control was 13.9 mg/100 g of body weight,4 whereas in the present experiment it was 15.5 mg/100 g of body weight. The subglomerulosa of W/KY animals was particularly prominent because of the virtual absence of lipid droplets. The function of this region is obscure, although mitosis occurs frequently in this region.' Our studies demonstrate that the cells have some characteristics of both neighboring zones: they have the same volume as those of the zona glomerulosa but increased volume of mitochondria and smooth endoplasmic reticulum, similar to those of the zona fasciculata. The cells in the subglomerulosa region may well represent transitional cells. Support for a transformation of zona glomerulosa cells into those of the zona fasciculata comes from the studies of Kahri." Newborn adrenocortical cells maintained in tissue culture have characteristics of the zona glomerulosa but become transformed into those with typical characteristics of the zona fasciculata upon addition of ACTH.
Refereles 1. Okamoto K, Aoki K: Development of a strain of spontaneously hypertensive rats. Jap Circ J 27:282-293, 1963 2. Okamoto K, Aoki K, Nosaka S, Fukushima NI: Cardiovascular diseases in the spontaneously hypertensive rat. Jap Circ J 28:943-952, 1964 3. Fregly MJ: Thyroid activity of spontaneous hy pertensive rats. Proc Soc Exp Biol Nied 149:124-132, 1975 4. Aoki K, Tankawa H, Fujinami T, Miyazaki A, Hashimoto Y: Pathological studies on the endocrine organs of the spontaneously hypertensive rats. Jap Heart J 4:426-442, 1963 5. Aoki K: Experimental studies on the relationship between endocrine organs and hypertension in spontaneously hypertensive rats. I. Effect of hypophvsectomv, adrenalectomv, thvroidectomv, nephrectomy and sympathectomy on blood pressure. Jap Heart J 4:443461, 1963 6. Rapp JP, Dahl LK: Adrenal steroidogenesis in rats bred for susceptibility and resistance to the hypertensive effect of salt. Endocrinology 88:32-65, 1971 7. NMoll 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, U lick S, Weinberger NIH: Control of aldosterone secretion in the spontaneously hypertensive rat. Circ Res 37:66-71, 1975 9. Taira Y: Histological and histoautoradiographical studies on the fetal adrenal cortex in spontaneously hypertensive rats. Acta Pathol Jap 24:733-746, 1974
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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, p 203 11. Maruyama T: Electron microscopic studies on the adrenal medulla and adrenal cortex of hypertensive rats. I. Spontaneously hypertensive rats. jap Circ J 33:1271-1284, 1969 12. Tsuchiyama H, Sugihara H, Kawai K: Pathology of the adrenal cortex in spontaneously hypertensive rats."" p 177 13. 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 Company, 1973, pp 237-296 14. Molteni A, Brownie AC, Skelton FR: Production of hypertensive vascular disease in the rat by methyltestosterone. Lab Invest 21:129-137, 1969 15. Nickerson PA, Brownie AC, Skelton FR: An electron microscopic studv of the regenerating adrenal gland during the development of adrenal regeneration hypertension. Am J Pathol 57:335-364, 1969 16. Stempak JG, Ward RT: An improved staining method for electron microscopy. J Cell Biol 22:697-701, 1964 17. 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 18. Nussdorfer GG: Effects of corticosteroid-hornones on the smooth endoplasmic reticulum of rat adrenocortical cells. Z Zellforsch Mikrosk Anat 106:143-154, 1970 19. Nussdorfer GG, Mazzocchi G, Rebuffat P: An ultrastructural stereologic study of the effects of ACTH and adenosine 3',5'-cyclic monophosphate on the zona glomerulosa of rat adrenal cortex. Endocrinology 92:141-151, 1973 20. Goldenberg MM: Study of cold plus restraint stress gastric lesions in spontaneouslv hypertensive, Wistar and Sprague-Dawley rats. Life Sci 12:519-527, 1973 21. 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 22. Bever KF, Samuels LT: Distribution of steroid-,B-ol-dehydrogenase in cellular structures of the adrenal gland. J Biol Chem 219:69-76, 1956 23. Ryan KJ, Engel LL: Hydroxylation of steroids at carbon 21. J Biol Chem 225:103-114, 1957 24. Brownie AC, Skelton FR: Adrenocortical function and structure in adrenal-regeneration and methyladrostenediol hypertension. Functions of the Adrenal Cortex. Edited by KW McKems. New York, Appleton-Centurv-Crofts, 1968, p 691 25. 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 26. Moses HL, Davis WW, Rosenthal AS, Garren LD: Adrenal cholesterol: Localization by electron microscope autoradiography. Science 163:1203-1206, 1969 27. Long JA, Jones AL: The fine structure of the zona glomerulosa and the zona fasciculata of the adrenal cortex of the opossum. Am J Anat 120:463-488, 1967 28. Deane HW, Shaw JH, Greep RO: The effect of altered sodium or potassium intake on the width and cytochemistry of the zona glomerulosa of the rat's adrenal cortex, Endocrinology 43:133-153, 1948 29. Hartroft PM, Eisenstein AB: Alterations in the adrenal cortex of the rat induced bysodium deficiency: Correlation of histologic changes with steroid hormone secretion. Endocrinology 60:641-651, 1957 30. Giacomelli F, Wiener J, Spiro D: Cytological alterations related to stimulation of the zona glomerulosa of the adrenal gland. J Cell Biol 26:499-522, 1965
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31. Long JA, Jones AL: Alterations in fine structure of the opossum adrenal cortex following sodium deprivation. Anat Rec 166:1-26, 1970 32. Kasemsri S, Nickerson PA: Quantitative ultrastructural studv of the rat adrenal cortex in renal encapsulation-induced hypertension. Am J Pathol 82:143-156, 1976 33. Koletskv S, Shook P, Rivera-Velez J: Lack of increased renin-angiotensin activitv in rats with spontaneous hypertension. Proc Soc Exp Biol Med 134:1187-1190, 1970 34. Brenner RM: Radioautographic studies with tritiated thymidine of cell migration in the mouse adrenal after a carbon tetrachloride stress. Am J Anat 112:81-95, 1963 35. Kahri A: Histochemical and electron microscopic studies on the cells of the rat adrenal cortex in tissue culture. Acta Endocrinol [Suppl 108] (kbh) 52:3-96, 1966
Acknowledgments The author is grateful to Neonile Fylypiw, Luther Joseph, Geneva Joseph, Elisabeth Lawson, and Robert Linsmair for skilled technical assistance. Berta Cole assisted with typing of the manuscript.
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[Illusatrao follow]
f-~
,
-
Figure 1-Zona glomerulosa cells from W/KY rat. Numerous lipid droplets (L) are observed in all of the zona glomerulosa cells. Mitochondria are circular to elongate in contour. Endoplasmic reticulum is predominantly smooth surfaced (ER). L Y = lysomsome, G = Golgi apparatus. ( x 16,000) Figure 2Cells in the subglomerulosa of W/KY animal. The cells are lipid free. Numerous cisternae of smooth endoplasmic reticulum (ER) are dispersed throughout the cell. Lysosomes (arrow) are numerous. G = Golgi apparatus, S = sinusoid. (x 16,000)
Figure 3-Zona glomerulosa cells from SHR. Many lipid droplets (L) are observed within the cells. Smooth endoplasmic reticulum (ER). One mitochondrion contains cristae which appear as straight tubules in section (arrow). (x 22,400)
Figure 4-Zona fasciculata cell from W/KY rat. Lipid droplets (L) are particularly numerous. Mitochondria are circular in contour and contain vesicular cristae. ER reticulum. (x 16,000)
=
smooth endoplasmic
3
4
5
Figure 5-Zona fasciculata cell from SHR. Smooth endoplasmic reticulum (ER) is scattered throughout Figure 6the cell. Mitochondrial cristae are tubulovesicular (arrow). L = lipid droplets. (x 22,400) The Zona fasciculata cells from SHR. Focal areas of glycogen (GL) are observed in the cytoplasm. Golgi apparatus (G) is particularly prominent. ER = smooth endoplasmic reticulum LY = lysosomes. (x 20,800)
