A Stereological Study of Adrenocortical Cells in Spontaneously Hypertensive Rats (SHR)

Path. Res. Pract, r62, 291-300 (1978)

Department of Pathology, University of Basle (Switzerland) and Department of Urology, University of Innsbruck (Austria)

A Stereological Study of Adrenocortical Cells in Spontaneously Hypertensive Rats (SHR) G. BARTSCH, U. BAUMGARTNER, and H. P. ROHR

Summary The ultrastructure of SHR (Spontaneously Hypertensive Rats) fasciculata cells was compared descriptively and quantitatively with that of nonstimulated Fasciculata cells of Wistar rats using stereological methods. The volume and surface densities are expressed per cm'' of cytoplasm. The smooth endoplasmic reticulum in the SHR was significantly increased compared to the control animals (surface density : 28%, volume density: 35%). Mitochondria volume remained unchanged although the inner mitochondrial membranes were significantly reduced (37010). An attempt was made to draw up a relation between stereological and biochemical data of steroid synthesis within the fasciculata cell. A genetically determined enzymatic defect in the early steps of the transformation of cholesterol to pregnenolone may exist at the level of the inner mitochondrial membrane. Whether this altered steroid metabolism is important to the etiology of hypertension in the SHR requires further Investigation.

Introduction Okamoto and Aoki (I) isolated a colony of rats in 1962 and 1963 which spontaneously developed hypertension with defined pathological lesions. They were called spontaneously hypertensive rats (SHR). In 1963 Aoki et al. (2) performed the first histological and histometrical study of the adrenal cortex of SHR and showed an increase in the zona fasciculata and zona reticularis as well as an increased lipid content of the cells in these Zones. Electron microscopic observations of the zona fasciculata of the SHR adrenal by Maruyama (3) indicated an increased secretory activity possibly due to chronic ACTH stimulation. Further data on the etiology and pathogenesis of this condition can be found in the review of Okamoto (4).

292 . G. Bartsch, U. Baumgartner, and H. P. Rohr

To elucidate a possible role of the adrenal cortex in the etiology of this hypertensive condition, more recently Moll et al. (5) studied steroid genesis by means of adrenal vein catheterization during the development of hypertension in SHR. The results (reduced secretion rates of 18-hydroxy-II-Bdeoxycorticosterone; I r-deoxycorticosterone; and corticosterone, the end product of steroid synthesis in the rat) suggested either an enzyme block or an increased conversion of known steroids to unknown steroid metabolites at the age of 20 weeks. The various cell components involved in steroid genesis have been successfully evaluated by stereological techniques, thereby providing quantitative data on the ultrastructure of the adrenal cortex under various experimental conditions (6-1 I, 19). An ultrastructural stereological study of the zona fasciculata of the adrenal gland in SHR was therefore performed in order to provide structural data complementary to the already existing biochemical work.

Material and Methods Five 200 ± I g male, adult Wistar rats, and three 210 ± 8 g male SHR F16 (4), were fed under rigorously standardized experimental conditions (Altromin-R standard diet, water ad libitum) and maintained on a regular day and night rhythm (12 : 12). On the basis of Okamoto's developmental study of the different periods of age we used 7 months old animals, thus choosing the interval between the early (4-6 months) and the advanced (12-14 months) hypertensive stage. Blood pressure was measured twice for each rat using the tail water plethysmographic method without anaesthesia. All 3 SHR were found to have sustained high systolic blood pressure; the means of the two measurements performed on SHR were as follows (animal 1,200 ± 5 mm Hg; animal 2, 180 ± 7.5 mm Hg; animal 3,190 ± 5 mm Hg). Electron microscopy

Tissue blocks (0.5-1 mm on a side) from one adrenal gland in each animal were fixed in I.33% osmium tetroxyde buffered with s-collidine (pH 7.4, total osmolarity mOsm) for IZO min at 4 '"CO The specimens were dehydrated by passage through increasing concentrations of alcohol followed by propylene oxide, and embedded in Epon (IZ). Sections (silver interference color) were cut with glass and in some cases with diamond knives on a Reichert ultramicrotome, mounted on zoo-mesh grids, and stained with uranyl acetate and lead citrate (lJ). Micrographs were made on 70 mm film plates with a Zeiss electron microscope EM 9A. The plates, together with corresponding test systems, were enlarged approximately 3.5 times and evaluated. The primary magnification was checked by including a micrograph of a calibration grating (Zeiss, Oberkochen, Germany) with each film plate batch. Stereologtcal analysis (Rohr et al., 1976)

For stereological analysis three blocks representing only tissue of the zona fasciculata were chosen at random and electron micrographs were taken at primary magnifications of 3,400 and 8,000. The sampling was done according to strict criteria of randomization.

Stereolog v of the Adrenal Cortex in Sl-l-Rats . 293

Fig . I. Zona Fasciculate reticulum (arrows).

10

SHR. Note the focal proliferation of smooth endoplasmic

Fig. 2. Detail of zona Fasciculara cell, showing a slight decrease of inner mitochondrial membranes. The smooth endoplasmic reticulum can be seen in a fine tubular as well as in a vesicular for m. Primary ma gni ficat ion A 8,0 0 0.

294 . G. Bartsch, U. Baumgartner, and H. P. Rohr For each block at least 10 electron micrographs were taken at both of these levels of magnification. A total of 60 micrographs was thus evaluated for each animal's zona [asciculara of the adrenal gland. For the purposes of the present study the fat droplets were analysed at a magnification of },400; and the mitochondria, the 5ER, the Goigi apparatus, the lysosomes and cytoplasmic ground substance at / 8,000. The cytoplasm was chosen as the basis of reference. The film plates were enlarged together with a multipurpose test screen (X },400) or a double square lattice system (. 8,000) and stereologically evaluated. Estimation of the volume density (V,) was given by the formula Vy = Pp where Pp is the fraction number of end point of the test lines enclosed within the profiles of the cell compartments. The evaluation of the surface density (5,,) of smooth endoplasmic reticulurn membranes was done by counting the number of intersections (I) of membrane profiles with the test lines by applying the formula Sv = 2 ;( IILT where LT is the length of the test lines enclosed in the cytoplasm. The results are expressed in m 2 per cm 3 of cytoplasm (14), For further methodological details see Rohr et al., (1976) (16).

Results Ultrastructural findings (Figs. I and 2) Fine structural changes in the cell or the zona fasciculata in the SHR were very striking and in general agreed with those found in previous reports (3, 4, 17)· The mitochondria did not show any alterations except a possible moderate decrease in the slightly flocculent matrix density. There was, as reported previously (3, 4, 17), an increase in smooth endoplasmic reticulum which sometimes showed a fine tubular configuration. Ultrastructural alterations of lysosomes and fat droplets were not observed.

Stereological findings Data of the various fasciculata cell compartments per unit volume of cytoplasm are given in Tables I and 2. The volume (11.40/0) as well as the surface density (2.5 m//cm") of the smooth endoplasmic reticulum were increased significantly in the SHR. The volume density and the surface density of the outer mitochondrial membrane remained unchanged; even the average volume of a single mitochondrium was not altered significantly (control, 1.26 urn"; SHR, 1.36 urn"). Conversely, the surface density of the inner mitochondrial membrane was significantly reduced in the SHR compared to controls (control, 5.8 m~!cm:J; SHR, 3.6 m 2/cm3) . The volume density of the lysosomes and the fat droplets was unchanged.

Discussion The stereological methods devised by Weibel (14) and Rohr, et al. (1976) (16) make possible a stereological analysis of the various cell com-

I.

Su rf acc

Surface

Volu me

Vo lume

Surface of outer membranes

Surface of inner membran es

Fat

Lysosomes V \'LY

VVF

Snw

Snro

V\'M

0.0 84 0 .00 8

cm 3/cm 3

5.88 I

m 2/cm 3 cm 3/cm3

1.694

0·374

em 3/em3 m ~/cm3

1.9P

0 .0 84

cm 3/em 3 m~/em3

C ontrol Mean

Unit

=

0.0°5

0.0°9

°'°78

0 .100

0.0 14

0 . 12

0.004

s.e.

(n

s.: significant, n.s. : not significant

The difference bewecn the Controls and the SHR is only considered to be significant if p

Volume

V\'SER

Surface

Mitochondria

VVi3ER

Symbol

Smooth endoplasmic reticulum Volume

P arameter

Va lue s per unit volume of Fasciculara cellu lar cytoplasm

Compartment

Table

<

3)

0.05

c.o ra

0.074

3.6 74

1. 87 8

0.4 14

2.5 04

0.114

SHR Mean

0.001

0.0 15

0. 2 55

0 .13 2

0 ·°3

c . 12 5

0 .006

(n = 3) s.e,

II. S.

n.s ,

s.

....., ° n.s,

V>

\0

l.>

:I: ~ ~

CJl

5'

§ '" ><

()

~

'" > 0.... '" ::s

s-

(JQ ~

n.s,

.... '"

~

0 '" 0

s.

s.

Significances

SYMo/NvM

Snrc!NVM

SYMc/V\')[

SYSERIVVSER

Surface of outer mitochondrial membranes

Surface of inner mitochondrial membranes

Surface-volume ratio

Surface-volume ratio

15. 684 23·3°8

m 2/cm3 m 2/cm3

=

2.02

0.63

0·547

0·353

C.081

s.e.

(n 5)

s.: significant, n. S.: not significant

The difference between the Controls and the SH-rats is only considered to be significant if p

19. 88

5.7 13

1. 265

fJ,m 2

flm2

flm 3

Vvu/Nva

Volume

Control Mean

<

0.05

21.9 12

8.872

12.173

6.22

1.3 67

SHR Mean

0.9 12

0.173

1.9 6

0.59 1

0.1°7

(n = 3) s.e.

n.s,

s.

s.

n.s,

n.s.

Significances

Values per average single mitochondrion and surface-volume ratio of mitochondria and smooth endoplasmic reticulum Unit

2.

Symbol

Table

l»

::r....

?=' o

~

~

0-

::l

~

(l)

.....

::l

~

"":4

"3

~

tl:l

~

~

:4

~

tl:l

o

0\

-o

Stereology of the Adrenal Cortex in SH-Rats . 297

partments at the ultrastructural level and allow a quantitative comparison of normal and experimentally altered tissue. The stereological data for smooth endoplasmic reticulum in the fasciculata cell of SHR is similar to that found during enhanced endogenous ACTH secretion or after exogenous ACTH administration (7, 9, 18). The volume and surface density of the SER was significantly increased. The values for the mitochondria were unchanged, except that the surface density of the inner mitochondrial membrane was significantly reduced. The ratio of the surface of the inner mitochondrial membranes to the volume density of the mitochondria shows that a real loss of inner mitochondrial membranes had occurred. The values for the volume densities of the fat droplets and the lysosomes did not change at all. A stereological analysis should help us to answer the question of whether steroid synthesis is reflected in the structural organization of the Fasciculata cell. Therefore, the biosynthesis of corticosteroids in the rat should be briefly summarized: According to Sayers et al. (20), steroid biosynthesis in the adrenal cortex involves a complex of enzymes within distinct compartments and it seems that a steroid molecule must travel a tortuous path back and forth among cellular organelles during the course of its transformation. Cell fractionation studies suggest that the enzymes involved in the conversion of cholesterol to pregnenolone are located in the mitochondria. Pregnenolome is transported out of the mitochondrion into the SER to be converted to progesterone, 17u-hydroxyprogesterone and r r-deoxycorticosterone. The hydroxylations at the positions 17 and 21 are confined to the SER (microsomal fraction). Hydroxylation at position I I (I I-~-hydroxylase) should take place in the mitochondrion, thereby forming the main glucocorticoid of the rat corticosterone (20). Recently, Moll et al. (5) indicated a markedly altered steroid synthesis in SHR, reflected by a lowered deoxycorticosterone and corticosterone secretion. The reasons for the reduced secretion rate of these adrenal gland steroids in SHR are unknown. Moll et al. (5) gave two possible explanations: (I) An enzyme block, resulting in decreased production of steroid end products with subsequent enhanced ACTH secretion or (2) an increased conversion of known precursors to other unknown steroids. In discussing the first possibility, that a genetic determined enzyme block is the cause of the hyperplasia of the adrenal cortex, the inhibition of steroid synthesis by metopirone blocking of the II-~-hydroxylase offers a good experimental model. Corticosterone in the rat participates in direct negative feedback with ACTH; lowered corticosterone secretion by blocking the I I-~-hydroxylase using metopirone leads to increased ACTH secretion.

29 8 . G. Bartsch, U. Baumgartner, and H. P. Rohr

As regards the smooth endoplasmic reticulum, the stereological data of Magelhaes and Magelhaes (9) and Rohr et al. (18) concerning the effects of metopirone administration on SER in metopirone-treated animals is very similar to data from SHR: In both conditions there is a marked increase in the volume and surface density of the SER, clearly demonstrating a proliferation of this cell compartment, which is most involved in the altered steroid synthesis. ACTH plays an important role in maintaining the functional integrity of the adrenal cortical cell compartments (7, 18, 19). According to the assumption of Moll et al. (5), that an enzyme block possibly is responsible for the failure of deoxycorticosterone and corticosterone in the SHR, the main steps of altered steroid synthesis in the SHR should take place in the SER. The demonstration of a proliferation of the SER smooth supports this hypothesis. The data of the mitochondria in the SHR did not show any alteration compared to the controls except that there was a high significant decrease in the surface density of the inner mitochondrial membranes. The loss of inner mitochondrial membranes could be best shown by examining the surface-to-volume ratio of this compartment. An opposite picture of an increased surface density in the inner and outer mitochondrial membranes was found upon inhibition of steroid synthesis by metopirone (Rohr et al., (18)). The enzymes (C-20, Cvza-desmolase, pregnenolone synthetase) involved in the conversion of cholesterol to pregnenolone are located in the mitochondria (20). One of the stimulatory actions of ACTH and of cyclicAMP has been established as the conversion of cholesterol to pregnenolone (22). This conversion involves a mixed-function oxidase system composed of mitochondrial cytochrome P 450 and a carrier protein (20). Yago et al., 1972 (19) demonstrated that the mitochondrial inner membranes contain P 450 cytochrome. In attempting to reconcile these established biochemical findings and our stereological data we might assume - thinking always of an enzyme block as the cause of altered steroid metabolism in SHR - that there is a genetically determined defect in the transformation of cholesterol to pregnenolone. Such an assumption may be an explanation for the reduced surface density of mitochondrial inner membranes in SHR, on the one hand, and for the increased surface density of inner mitochondrial membranes following metopirone administration, on the other hand. Unfortunately, Moll et al. (5) did not measure pregnenolone production, which might have indicated an earlier enzyme block in the adrenal cortex in the SHR. This stereological data possibly gives us a new insight into the ultra-

Stereology of the Adrenal Cortex in SH-Rats . 299

structural changes which have been observed previously in SHR. Whether or not the altered steroid metabolism or the increased levels of ACTH are important to the etiology of hypertension in SHR is not clear (24). The stereological findings possibly indicate that the adrenal gland constitutes an important factor in producing spontaneous hypertension in this type of rat (5).

References I.

2.

3. 4. 5. 6. 7.

8. 9. 10.

1I.

12. 13. 14. 15. 16. 17.

Okamoto, K., and Aoki, K.: Development of a strain of spontaneously hypertensive rats. Circulation J. 27 (Full. Ed.), 282-293 (1963) Aoki, K., Tankawa, H., Fujinami, T., Miyazaki, A., and Hashimoto, Y.: Experimental studies on the relationship between endocrine organs and hypertension in spontaneously hypertensive rats. Jap. Heart J. 4,5 6 1-576 (1963) Maruyama, T.: Electron microscopic studies on the adrenal medulla and adrenal cortex of hypertensive rats. Circulation J. 33 (Jap.), 1271-1278 (1969) Okamoto, K.: Spontaneous Hypertension in Rats. International Review of Experimental Pathology 7, pp. 227-27°. Academic Press, New York-London (1969) Moll, D., Dale, S. L., and Melby, J.: Adrenal Stereoidogenesis in the Spontaneously Hypertensive Rat (SHR). Endocrinology 96, 4 16-4 20 (1975) Nussdorfer, G. G.: Effect' of Corticosteroid-Hormones on the Smooth Endoplasmic Reticulum of Rat Adrenocortical Cells, Z. Zellforsch. lO6, /43-/54 (/970) Nussdorfer, G. G., and Mazzocchi, G.: A Stereologic Study of the Effects of ACTH and Cyclic 3',(-AMP on Adrenocortical Cells of Intact and Hypophysectomized Rats. Lab. Invest. 26, 45-52 (1972) Canick, A. ]., and Purvis j. L.: The Maintenance of Mitochondrial Size in the Rat Adrenal Cortex Zona Fasciculata by ACTH. Exp. Molec. Path. /6, 79-93 (1972) Magelhaes, M. c., and Magelhaes, M. H.: A Stereologic Study of the Effects of Metopiron on the Rat Adrenal. Lab. Invest. 21, 491-496 (1969) Kahri, A. 1.: Selective Inhibition by Chloramphenicol of ACTH induced Reorganization of Inner Mitochond r ral Membranes in Fetal Adrenal Cortical Cells in Tissue Cultures. Amer. J. Anat. 127, 101-13° (1970) Rohr, H. P., Bartsch, G., Eichenberger, P., Rasser, Y., Kaiser, Ch., and Keller, M.: Ultrastructural Morphometric Analysi, of the Unstimulated Adrenal Cortex of Rat. J. Ultrastr. Res. 54, 11--21 (1976) Luft, J. H.: Improvement, In Epoxy resin Embedding Methods. J. Biophys. Cytol. 9,4°9-418 (19 6 1) Reynolds, E. R.: The Use of lead Citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell. Biol, 17, 208-212 (1963) Weibel, E. R.: Stereological principles for morphometry in electron microscopic cytology. Int. Rev. Cytol, 27, 235-302 (1969) Weibel, E. R., and Gomez, D. H.: A principle for counting tissue structures in random sections. J. appl. Physiol. 17, 343-354 (1967) Rohr, H. P., Oberholzer, M., Bartsch, G., and Keller, M.: Morphometry in Experimental Pathology. Int. Rev. e"p. Path. 15,233-325 (1976) Tabei, R., Naruyama, T., Kumuda, M., and Okamoto, K.: In Okamoto, K. (Ed.): Spontaneous Hypertenvion, pp. 185-196. Igaku Shoin Ltd., Tokyo (1972)

30 0

.

G. Bartsch, U. Baumgartner, and H . P. Rohr

18. Rohr, H. P., and Bart sch, G.: Inh ibition of 11-f3-hydro xylase by metopirone (Stereo logical anal ysis), in pr epara tion, 1978. 19. Yago, M., Seki, M., Sekiyama, 5., Koba yashi,S., Kurokawa, H ., Iwai , Y., Saw, F., and Shiragai, A.: Growth and Differentiation of Mitochondr ia in the regenerating rat adrenal corte x. J. Cell. BioI. 52,5 ° 3-513 (1972) 20. Sayers, G., Beall, R., and Seelig, 5.: Modes of Action of ACTH in MTP. International Review of SCience, Biochemistr y of Ho rmo nes, pp . 26-5 0. Butterworth, London (1974) 21. Gaunt, R., Chan, J. j. . and Renzi, A. A.: Inhibitors of Adr enal Cortical Function. Rev. Ph ysio!. Biochem, expoPharm ac. 56, 114-1 72 (1965) 22. Karaboyas, G. c., and Kontz. S. B.: Identity of the site of act ion of J' ,5'-adenosine monophosphare and ad renoco rtico tr opic hormone in cort icosteroidogenesis in rat adrenal and beef adren al cortex slices. Biochemistry 4, 462-468 (1965) 23. Boyd, G. 5., Brownie, A. C; and j efcoare, C. R. : Cholesterol hydroxylation in the ad renal cortex and liver. Biochem. J. 125, 19-29 (1971) 24. Baes, L., Knowton, A.• and l.a ragh, J. H.: In Okamoto, K. (Ed.): Spontaneous Hypertension, pp. 2°3-242. Igaku Shoin Ltd .• Tokyo (1972)

Received October 17, 1976 ' Accepted in revised form February 2,1978

Key w ords: Adrenal cortex - Zona fasciculata - Electron microscopy Stereo logy - Steroid Synthesis - Mitochondria - Smooth Endoplasmic reticulum - Hyperten sion H . P. Rohr, M. D., Institute of Path ology, Schonbeinstr. 40, 4056 Basle, Switzerland