ULTRASTRUCTURE OF THE ADRENAL CORTEX IN FROZEN THIN SECTIONS

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1 J. Cell Set. 27, 3C3-3" (i977) 303 Printed in Great Britain Company of Biologists Limited ULTRASTRUCTURE OF THE ADRENAL CORTEX IN FROZEN THIN SECTIONS M. M. MAGALHAES Laboratory of Histology and Embryology, Faculty of Medicine, Porto, Portugal SUMMARY Pieces of both unfixed and fixed rat adrenal cortex were frozen and cut with a cryoultramicrotome. In unstained frozen sections of unfixed adrenals patches of heterochromatin were seen in the nucleus, while numerous round uniformly dense structures, the mitochondria, could be identified in the cytoplasm. Lipid droplets appear as small light round areas. Staining the sections with potassium permanganate, phosphotungstic acid, or uranyl acetate, or immersing them in glutaraldehyde, increased the contrast of cell organelles. In frozen sections of fixed material, membranes became visible in negative contrast. Mitochondria display vesicular cristae, whereas the Golgi complex, lysosomes and peroxisomes are easily recognizable. The possibility of cutting frozen sections of unfixed material with reasonable morphological preservation allows the fine-structural localization of steroids in adrenal cells after applying autoradiographic emulsion to the sections. INTRODUCTION Although attempted by a few workers (Idelman, 1968; Moses, Davis, Rosenthal & Garren, 1969; Magalhaes & Magalhaes, 1974), the identification of the sites of synthesis, conversion and migration of adrenal cortex hormones in undisrupted cells is fraught with great difficulties. Fixation and dehydration procedures used for electron microscopy and conventional ultrastructural autoradiography are liable to cause extraction and displacement of steroid hormones (Idelman, 1968; Moses et al. 1969). The only way to circumvent these drawbacks would be to use pieces that have had no contact with any liquid during the preparation of the material for autoradiography. Therefore knowledge of the ultrastructural features of the adrenal cortex in frozen thin sections obtained under different experimental conditions is of the utmost interest. This paper describes the fine structure of adrenal cells as shown in thin frozen sections of unfixed and fixed material. An attempt at performing autoradiography with such sections is described. MATERIALS AND METHODS Small pieces of fresh adrenals were removed from anaesthetized male rats (Sprague-Dawley) approximately 200 g in weight. Under the dissecting microscope the pieces were cut into pieces and placed on the end of a chuck, frozen against a copper block (Christensen, 1971) previously brought to liquid nitrogen temperature, and stored in liquid nitrogen until sectioning.

2 304 M. M. Magalhaes Thin frozen sections were prepared (Christensen, 1971) in an MT2-B Porter Blum, ultramicrotome with the Sorvall-Christensen FTS-LTC-2 automatic frozen thin sectioner system, at 70 C. The sections were picked up from the glass knife edge with an eyelash probe, placed on Formvar/copper grids and flattened with a copper rod. The grids were left for some time in the bowl for drying (Christensen, 1971). After the sections were dry, they were stored in a vacuum desiccator or observed immediately in the electron microscope (Philips EM 300) operated at 60 kv. Grids were examined unstained or after staining. The following staining procedures were carried out: (a) 2% phosphotungstic acid (PTA) for 30 s (Bernhard & Viron, 19.71); (b) 0-5 % uranyl acetate in distilled water for 5 min (Bernhard & Viron, 1971); and (c) 1% potassium permanganate for 30 s (Sutton, 1968). Some grids were contrasted with glutaraldehyde. In this case dried sections were immersed in 2 % glutaraldehyde in o-i M phosphate buffer for 30 min and washed in distilled water. After glutaraldehyde treatment the grids could also be stained with PTA or uranyl acetate as described above. Some adrenal pieces were fixed in 2 % glutaraldehyde in o-i M phosphate buffer and treated according to the procedure of Tokuyasu (1973). The adrenal pieces were infused with 1-5 M sucrose in O'i M phosphate buffer for 30 min, placed on the chuck and immersed directly in liquid nitrogen. Thin frozen sections were cut at 70 C, mounted on the grids as described by Tokuyasu (1973), stained or not with PTA, and observed in the electron microscope. For autoradiography the grids with dried sections were placed inside the microtome bowl in a small box in which vacuum was made; the box with the sections was transferred to the evaporator where the sections were carbonized. Then the emulsion was applied directly on to the sections. Contrast was enhanced by staining the autoradiographs with glutaraldehyde after the autoradiographic procedure. RESULTS In unstained frozen sections of unfixed pieces no membrane outlines are visible but the main organelles of the adrenal cell can be recognized due to differences in contrast (Fig. 1). Thus, the periphery of the nucleus is marked by heavy patches of a material (Fig. 1) which by comparison with routinely fixed and stained pieces can be identified as condensed chromatin. In the cytoplasm, round dense masses, fim in diameter can be recognized by their number and distribution as mitochondria (Fig. 1). Between them, there are spaces of medium density in which the profiles of endoplasmic reticulum are not discernible (Fig. 1). Small round areas devoid of any density but with regular contour seem to represent lipid droplets. Staining of those sections with potassium permanganate enhanced the overall density of mitochondria but it did not reveal mitochondrial membranes or the membranes of any other structure (Fig. 2). Staining with phosphotungstic acid or uranyl acetate caused the same effects. Immersion of the sections in glutaraldehyde markedly enhanced the density of chromatin and rendered the contours of chromatin and lipid droplets sharper (Fig. 3). Frozen sections of glutaraldehyde-fixed material allow a considerable improvement in morphological detail. Membranes are clearly denned in negative contrast, so that it is possible to follow the outline of most cell components. The nuclear envelope with its 2 membranes and the perinuclear cisternae are observed. Mitochondrial membranes, either vesicular or tubular, stand out against the dark matrix (Figs. 4, 5), and matrix inclusions made up of parallel dense bands separated by white spaces are observed at high magnification (Fig. 6). Lysosomes, peroxisomes, as well as

3 Ultrastructure of adrenal cortex 3 5 Fig. 1. Frozen thin sections of the zona fasciculata of unfixed adrenal cortex. The picture shows 2 nuclei (n) with heterochromatin patches. Numerous mitochondria (m) are observed in the cytoplasm. Regular holes of medium size may correspond to lipid droplets (/). Larger irregular holes ( ) are probably artifactual. Unstained section, x Fig. 2. Frozen sections of the same material as in Fig. 1, but with the section stained with potassium permanganate. Numerous mitochondria (w) are seen in the cell cytoplasm, x

4 306 M. M. Magalhaes tubular or vesicular elements of the endoplasmic reticulum, are apparent in the intermitochondrial spaces. Finally, stacks of Golgi cisternae are frequently seen near the nucleus (Fig. 5) or at the cell periphery. Fig. 7 shows the result of applying autoradiography to frozen sections of unfixed material. Fig. 3. Same material as in Fig. 1. The section was immersed in glutaraldehyde for 30 min. I, lipid droplets; n, nucleus, x DISCUSSION The subcellular localization of adrenal steroids has been attempted in recent years. Using the current techniques of electron-microscope autoradiography, Moses et al. (1969) were able to localize most of the adrenal cholesterol in lipid droplets, while Magalhaes, Magalhaes & Coimbra (1974) observed that conversion of deoxycorticosterone (DOC) into corticosterone apparently took place in adrenal mitochondria in vitro and that accumulation of exogenous DOC occurred in lipid droplets when 11 /?-hydroxylation was prevented. Although these authors have employed fixation and dehydration procedures designed to maximize steroid retention, the use of numerous solvents in tissue preparation and through the autoradiographic technique could still be a cause of removal or displacement of steroids. In fact, Idelman (1971) showed that Epon embedding extracted % of the adrenal radioactivity after pregnenolone administration. Hence, in any electron-microscope study of the sites of synthesis, conversion

5 Ultrastructure of adrenal cortex 307 Fig. 4. Frozen section of the zona fasciculata of rat adrenal fixed with glutaraldehyde. Sections were prepared according to the method of Tokuyasu (1973). The picture shows numerous mitochondria (tri) with vesicular cristae and the endoplasmic reticulum (er) composed of vesicular elements. All the membranes are negatively stained. /, lipid droplets; p, peroxisomes. Section stained with PTA. x

6 3o8 M. M. Magalhaes Fig. 5. Frozen section of the zona glomerulosa of rat adrenal fixed with glutaraldehyde. Mitochondria (m) exhibit tubular cristae; in the rest of the cytoplasm vesicular and tubular membranous profiles of the endoplasmic reticulum (er) are present; near the nucleus («), elements of the Golgi complex (go). Section stained with PTA. x Fig. 6. Frozen section of fixed adrenal. Detail of a mitochondrion of the zona fasciculata. Observe 2 paracrystalline inclusions with fine structure similar to that found in adrenal sections in Epon. Staining as in Fig. 4. x

7 Ultrastructure of adrenal cortex 309 Fig. 7. Autoradiograph of a frozen section of the zona fasciculata of unfixed adrenal cortex. Note the silver grains over the mitochondria (m) and the endoplasmic reticulum spaces; n, nucleus. Incubation of adrenal slices in medium with 200 /tci of PH]cholesterol for 3 min. Unstained section, x CEL 27

8 310 M.M. Magalhaes and migration of steroids, it is essential that the tissue should not come into contact with any solvent. At the light-microscope level, Stumpf & Roth (1966) showed that the tissue must be frozen without fixation, the material must be maintained below 60 C during sectioning and freeze-drying, and finally that contact with any fluid should be avoided until the autoradiographic process is completed. The first problem in localizing adrenal steroids in electron microscopy thus consists of cutting frozen thin sections of unfixed tissue. The preparation of frozen sections of unfixed adrenals, however, presents some problems. It is difficult to cut frozen thin sections on account of ice crystal formation during freezing and later recrystallization. This is perhaps due to the high water content of adrenal cells which makes vitrification difficult (Rebhun, 1972). Nevertheless it was possible for us to obtain sections of tissue areas which, though poor in morphological detail, allowed a clear demarcation of nuclei, mitochondria, and spaces of the endoplasmic reticulum. Since these 3 compartments are the sites of the main metabolic steps of steroidogenesis, identification of the areas occupied by them may be of great value in applying autoradiography to this material. As a matter of fact, the resolution of electron-microscope autoradiography is not fine enough to detect radioactive sources of silver grains in the inner components of mitochondria, in particular cisternae of the endoplasmic reticulum, or in other similarly detailed structures. It can, however, be used in locating radioactive reactions over whole mitochondria or endoplasmic reticulum spaces. Contrast is enhanced by subsequent treatment of frozen sections with glutaraldehyde or by uranyl acetate staining. This is understandable, because proteins are cross-linked with glutaraldehyde, while DNA, proteins and phospholipids react with uranyl ions during staining (Hayat, 1970). However, such treatments may extract or displace steroids, so that they are of no use in locating such substances. Frozen sections of fixed adrenals are easier to cut and visualization of the ultrastructure is considerably improved. The nucleus shows distinct patches of condensed chromatin and the nucleolus appears discrete, the details of mitochondrial structure are clearly revealed, though the membranes are negatively stained. Intramitochondrial paracrystalline inclusions similar to those observed in adrenals prepared by the conventional electron-microscope technique (Magalh2es & MagalhSes, 1968; Fruhling, Meneghelli 8c Claude, 1968) were also visible. This procedure may be promising in localizing enzyme activities as well as substances that are not extracted or displaced by the fixatives, since sections without plastic embedding permit the easy access of large molecules to chemically reactive groups (Bernhard & Viron, 1971). However, the use of glutaraldehyde for fixation and of sucrose solutions for tissue infusion are again not compatible with steroid localization. In conclusion, the only procedure that may allow the identification of steroids in situ is the use of frozen sections of unstained material, eventually coupled with dry autoradiography (Christensen & Paavola, 1972). This work was carried out while the author was a visiting investigator (fellow of the Instituto de Alta Cultura, Lisbon, Portugal) in the laboratory of Dr A. Kent Christensen, Department of Anatomy, Temple University School of Medicine, Philadelphia, Pennsylvania, U.S.A., from

9 Ultrastructure of adrenal cortex 311 November 1972 to October I want to thank Dr Christensen and Dr Laurie G. Paavola for making available their technique of preparing frozen thin sections and their method of carrying out frozen thin-section autoradiography, as well as for help and advice during my year in that laboratory. REFERENCES BERNHARD, W. & VIRON, A. (1971). Improved techniques for the preparation of ultrathin frozen sections. J. Cell Biol. 49, CHRISTENSEN, A. K. (1971). Frozen thin sections of fresh tissue for electron microscopy with a description of pancreas and liver. J. Cell Biol. 51, CHRISTENSEN, A. K. & PAAVOLA, L. G. (1972). Frozen thin sections of fresh tissue and their possible use for autoradiography of diffusible substances. Ada histochem. cytocliem. 5, FROHLING, J., MENECHELLI, V. & CLAUDE, A. (1968). Inclusions tubulaires a organisation paracristalline et leurs rapports avec les crfites dans les mitochondries de la corticosurrenale du rat et du boeuf. J. Microscopie 7, HAYAT, M. A. (1970). Principles and Techniques of Electron Microscopy. Biological Applications, vol. 1, 1st. edn, pp New York: Van Nostrand Reinhold. IDELMAN, S. (1968). Preservation des st^roides de la glande cortico-surrenale du rat au cours de la fixation puis de l'inclusion dans l'6pon. C. r. hebd. Sianc. Acad. Set., Paris 266, IDELMAN, S. (1971). Application de l'ultracryostat a l'observation infrastructurale du cortex surr^nal du rat. Etude des conditions d'application a l'autoradiographie de pr^cureeurs hormonaux. C. r. du Coll. In Ultrastructure des Cellules a Secretion de Stiroides, pp Paris: Clermont-Ferrand. MAGALHAES, M. C, MAGALHAES, M. M. & COIMBRA, A. (1974). An electron microscope autoradiographic study of DOC conversion to corticosterone in the rat adrenal cortex. Cell Tiss. Res. 151, MACALHSES, M. M. & MAGALHAES, M. C. (1968). Inclusions intramitochondriales a structure cristalline dans la cortico-surrenale du rat. J. Microscopie 7, MOSES, H. L., DAVIS, W. W., ROSENTHAL, A. S. & GARREN, L. D. (1969). Adrenal cholesterol: localization by electron-microscope autoradiography. Science, N.Y. 163, REBHUN, L. T. (1972). Freeze-sub8titution and freeze-drying. In Principles and Techniques of Electron Microscopy. Biological Applications, vol. 2 (ed. M. A. Hayat), istedn, pp New York: Van Nostrand Reinhold. STUMPF, W. E. & ROTH, L. J. (1966). High resolution autoradiography with dry mounted, freeze-dried frozen sections. Comparative study of six methods using two diffusible compounds 3 H-estradiol and 3 H-mesobilirubinogen. J. Histochem. Cytochem. 14, SUTTON, J. S. (1968). Potassium permanganate staining of ultrathin sections for electron microscopy. J. Ultrastruct. Res. 21, TOKUYASU, K. T. (1973). A technique for ultracryotomy of cell suspensions and tissues. J. Cell Biol. 57, 55I-565- {Received 8 December 1976)