Silver-Impregnation of the Golgi Complex in Epididymal Epithelial Cells of Mice

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1 CELL STRUCTURE AND FUNCTION 8, (1984) C by Japan Society for Cell Biology Silver-Impregnation of the Golgi Complex in Epididymal Epithelial Cells of Mice Ikuo Yamaoka, Sumie Katsuta and Yoshimi Nagatani Biological Institute, Faculty of Science, Yamaguchi University, Yamaguchi 753, Japan ABSTRACT. Localization of silver grains detected by the silver-impregnation method, a technique used to detect the classical Golgi apparatus, was examined with light and electron microscopy. Two types of silvered images of the Golgi apparatus were compared; each was obtained by Da Fano's silverimpregnation method, and one was modified with Caulfield's fixative during the preliminary fixation. Under ordinary light microscopy the images were very similar and showed the duplex structure of the Golgi apparatus which consists of an argentophil wall and argentophobe core. With electron microscopy, the relationship between the fine structure of the Golgi complex and the silver deposits was obtained in greater detail by the latter technique because the fine structure of the Golgi complex was retained. Many fine silver grains were detected in the cytoplasm adjacent to the Golgi complex, but none were present in the Golgi cisternae. This suggests that the argentophil wall of the duplex structure of the classical Golgi apparatus may be formed from argentophil substances that locate in the cytoplasm adjacent to the Golgi lamellae, and that the argentophobe core may be related to the Golgi lamellae. Since the use of osmium-impregnation as a cytological marker for the Golgi complex in electron microscopy was introduced by Dalton and Felix (5, 6), several reports on the distribution of osmium deposits in the Golgi complex have appeared (11, 12, 20). But there has been little reported of electron microscopical studies on the distribution of silver grains obtained by the silver-impregnation technique. Lacy and Challice (16) reported that silver grains were detectable in the cytoplasm adjacent to the Golgi complex, but in their electron micrographs no details of the distribution of the silver grains could be seen because the fine structure of the cell, especially that of the Golgi complex had not been well retained. One cause of this lack of retention may be the fixative used. In the classical technique, usually unbuffered fixatives have been used, as in Aoyama's, Da Fano's and Elfman's methods. We have obtained clear distributions of silver grains by using a modified silver-impregnation technique and electron microscopy. Both our present and previous results (22) with osmium-impregnation agree in locations, especially location of fine particule deposits. Thus, the similarities in location of the silvered and osmicated images of the classical Golgi apparatus could be confirmed by electron microscopy. 339

2 340 I. Yamaoka, S. Katsuta and Y. Nagatani MATERIALS AND METHODS Three month-old mice (dd strain) were purchased from a dealer, and maintained at 25 Ž until required. The middle segment of the anterior epididymis of each mouse was cut out and fixed at 4 Ž for 3 h in Da Fano's (4) or Caulfield's (2) fixatives (ph 7.4) with 4.5 % added sucrose. Da Fano's fixative was combined with NaHCO3 (0.8 mg/ml original solution) according to Motomura's modification (personal communication). After fixation specimens were immersed at 25 C for 3, 6, 12, and 24 h in a 1.5 % aqueous solution of silver nitrate in the dark. In Da Fano's method immersion was for 12 h only. After immersion the samples were washed with distilled water then reduced for 6 h in Cajahl's solution, after which they were dehydrated in the ethanol and acetone series then embedded in Epok 812. For light microscopy thick sections were placed on a slide glass and stained with a heated 1 % aqueous solution of methylene blue at a high ph. Ultrathin sections used for electron microscopy were stained with aqueous uranyl acetate and lead citrate. In some instances these sections were stained with lead citrate only. The prepared sections were examined with a JEM-100C electron microscope (JEOL. Ltd.). RESULTS Silvered image of the Gogi apparatus obtained by Da Fano's method. Under light microscopy the silvered image of the Golgi apparatus in epididymal epithelial cells of the mouse showed up as a massive body located in the supranuclear region in the columnar cells. Under high magnification (Fig. I a), however, this massive body appeared as a reticular aggregate composed of successive granules of various size and form. Electron microscopy showed that these granules were composed of condensed fine silver grains, on both sides of the large vacuole (Fig. lb). The relation between the silver grains and the organelles was not clear because the fine structure of the cell was not well retained. Classical Golgi apparatus obtained by our modified method. After fixation in Caulfield's fixative the epithelial tissue was immersed for various periods in an aqueous solution of silver nitrate. After a short exposure (3 h), extensive deposits of reduced silver which had no relationship to the special cytoplasmic architecture were found throughout the cytoplasm (Fig. 2a). After prolonged exposure (more than 6 h), a specific distribution of silver deposits appeared in the supranuclear region (so-called Golgi area). Our silvered images of the Golgi area for the 6- and 12-h preparations showed a thin reticular form composed of a succession of granules (Fig. 2b and 2c). This reticular structure was clearer along its periphery than at the center of the Golgi area. A granular condensation usually was present inside the Golgi area. The duplex structure of the Golgi apparatus, which consists of an argentophil wall and argentophobe core, was very clear in the 12-h preparation (Fig. 3a). Twenty four-hours after commencement of treatment silver deposits were scattered throughout the cell apex and were present in the intercellular space (Fig. 2d). Fig. 1. Light (a) and electron (b) micrographs 12 h after Da Fano's silver-impregnation. (a): reticular or massive blackened granules are present in the supranuclear region. Granules vary in size and form, some surround a small vacuole and form a duplex structure (arrow). (b): Electron micrograph of the same preparation seen in (a). Large vacuoles are present in the supranuclear region, on both sides of which are many silver grains. The fine structure of the cell has not been well retained. N, nucleus. V, vacuole. B 1, basal lamina. a, ~ b, ~ 6000.

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5 Silver-Impregnation of the Golgi Complex 343 Ultrastructure of the Golgi complex in normal and silvered specimens. The fine structure of the Golgi complex in epididymal epithelial cells from normal mice has been described recently (22). In brief, it is well developed and located in the supranuclear region. It is circular or semicircular in form and consists of ten or more parallel lamellae. Many vesicles containing moderately electron-dense materials were seen near the Golgi lamellae and many more on its inner concave face. The fine structure of the cell in silver-impregnated (12 h) specimens was well retained (Fig. 3b), and the Golgi complex was similar to that of normal specimens. Fine silver grains were present on both sides of the stacked Golgi cisternae, but none were found inside the cisternae. A massive deposition, partially outside the Golgi lamellae, was present, and each mass was composed of many fine deposits (Fig. 3b). In the light micrograph the massive silvered deposits were located specifically in the Golgi area (Fig. 3a). The duplex structure also was clearly delineated (Fig. 3a, arrow). DISCUSSION During the last 30 years much information about the Golgi complex has been obtained by the use of autoradiographic, cytochemical and biochemical techniques. The major histrorical events of the development of this knowledge have been detailed by Farquhar and Palade (7). Many studies of the organization of the Golgi complex have shown that the whole structure has a clearly recognizable polarity and that there is compositional heterogeneity among the Golgi cisternae. The latter results comes from cytochemical staining of a marker enzyme, first reported by Novikoff and Goldfischer (19) and later by several researchers (3, 8, 12, 21). Subsequent studies of marker enzymes combined with the fractionation of the Golgi cisterna have provided new information on the function of the Golgi complex (1, 3, 8, 9, 10, 17). The study of the substance surrounding the Golgi complex, has remained a problem, but metal impregnation is known to be a key procedure verifing the details of the Golgi apparatus. We have investiagted interactions between the Golgi complex and the substance that surrounds this complex. Since the introduction of osmium-impregnation as a marker of the classical Golgi apparatus in electron microscopy by Dalton and Felix (5, 6), this technique has gained wide use in studies of various cell types (11, 12, 13, 15, 20). Little use of the silver-impregnation method, however, has been made in studies of the classical Golgi apparatus. Lacy and Challice (16) found silver grains deposited on one or both sides of vacuoles located within the Golgi zone in pancreatic acinar cells. The fine structure of the Golgi apparatus was not, however, retained in good condition. Probably this was Fig. 2. Light micrographs of the silvered image formed by Caulfield's fix/silver-impregnation method. (a): 3 h after silver-impregnation blackened granules were detectable at the interfaces and in the apical portions of the cells. No specific localization in the Golgi area is visible yet. (b) : 6 h after silver-impregnation a continuous arrangement of silver granules is present in the supranuclear region. Some granules have a thin reticular form (arrow). (c): 12 h after silver-impregnation the silvered image of the Golgi complex is similar to that in (b), but the duplex structure is clear (arrow). (d): 24 h after silver-impregnation the massive silvered image in the supranuclear region has become small; many granules are also present in the apexes and at the interfaces of the cells as in (a). N, nucleus. L, lumen. ~ 660.

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7 Silver-Impregnation of the Golgi Complex 345 because they used Aoyama's method that employs a fixative for silver-impregnation that is unsuitable for electron microscopy. Previously, we used Da Fano's method, as modified by Motomura, to detect the classical Golgi apparatus in epididymal epithelial cells of mice and obtained a typical image of the Golgi apparatus (18). We now have obtained similar results with light microscopy, but with electron microscopy our results were similar to those of Lacy and Challice (16); that is, the silver deposits were distributed near the large vacuole in the Golgi area. The problem of retaining the fine cell structure when the silver-impregnation technique is used was resolved by using Caulfield's fixative in the preliminary fixation. As a result, the silvered image in the Golgi area showed a reticular form and that resembled the discription given by Nagatani and Yamaoka (18). Thus, the blackened image we have obtained can be regarded as that of the classical Golgi apparatus. Electron microscopy showed that the silver grains visualized by the new method were located in the cytoplasm on both sides of the Golgi complex, but none were present in the Golgi cisternae. There are a number of reports on different types of cells that show that the distribution of reduced osmium is limited to one or two layers of the Golgi lamellae or to vesicles of the transitional elements (11, 12, 13, 14, 20). Recently, Yamaoka et al. (22) reported that the distribution of deposits of reduced osmium in epididymal epithelial cells of the mouse could be divided into groups ; deposits in the Golgi cisternae and in the cytoplasm adjacent to the Golgi complex. The latter deposits were particular and especially abundant during the recovery stage of hormone treatment after castration. The distribution of silver grains which we obtained was similar to that for particular deposits of reduced osmium. We then confirmed the similarity between the silvered and the osmicated images of the classical Golgi apparatus by electron microscopy. Although the reason for the deposition on both sides of the Golgi complex has yet to be determined, the true appearance of the classical Golgi apparatus can be assumed to be caused by some argentophil or osmiophil substance that is located in the cytoplasm adjacent to the Golgi complex. REFERENCES 1. BERGERON, J.J.M., J.H. EHRENREICH, P. SIEKEVITZ and G.E. PALADE. Golgi fractions prepared from rat liver homogenate. II. Biochemical characterization. J. Cell. Biol. 59, 73-88, CAULFIELD, J.B. Effects of varying the vehicle for 0504 in tissue fixation. J. Biophys. Biochem. Cytol. 3, , CHENG, H. and M.G. FARQUHAR. Presence of adenylate cyclase activity in Golgi and other fractions from rat liver. II. Cytochemical localization within Golgi and ER membranes. J. Cell Biol. 70, , DA FANO, C. Method for the demonstration of Golgi's internal apparatus. J. Physiol. 53, 92-94, DALTON, A.J. and M.D. FELIX. Cytological and cytochemical characteristics of the Golgi Fig. 3. Light (a) and electron (b) micrographs 12 h after Caulfield's fix/silver-impregnation. The specific distribution of silver grains in the Golgi area is shown (a). (b): The fine structure of the Golgi complex has been well retained. Fine silver grains are located on both sides of the Golgi lamellae, but there are none in the Golgi cisternae. GA, Golgi area. GL, Golgi lamellae. a, ~ b, ~ 6000.

8 346 I. Yamaoka, S. Katsuta and Y. Nagatani substance and epithelial cells of the epididymis-in situ, in homogenates and after isolation. Am. J. Anat. 94, , DALTON, A.J. and M.D. FELIX. A comparative study of the Golgi complex. J. Biophys. Biochem. Cytol. 2 (suppl.), 79-84, FARQUHAR, M.G. and G.E. PALADE. The Golgi apparatus (Complex) -( )- from artifact to center stage. J. Cell. Biol. 91, 77s-103s, FARQUHAR, M.G., J.J.M. BERGERON and G.E. PALADE. Cytochemistry of Golgi fractions prepared from rat liver. J. Cell Biol. 60, 8-25, FLEISCHER, B. and S. FLEISCHER. Preparation and characterization of Golgi membarnes from rat liver. Biochem. Biophys. Acta 219, , FLEISCHER, D., S. FLEISCHER and H. OZAWA. Isolation and characterization of Golgi membarnes from bovine liver. J. Cell Biol. 43, 59-79, FRIEND, D.S. Cytochemical staining of multivesicular body and Golgi vesicles. J. Cell Biol 41, , FRIEND, D.S. and M.J. MURRAY. Osmium impregnation of the Golgi apparatus. Am. J. Anat. 117, , HAND, A.R. Cytochemical differentiation of the Golgi apparatus from GERL. J. Histochem. Cytochem. 28, 82-86, HAND, A.R. and C. OLIVER. Relationship between the Golgi apparatus GERL, secretory granules in aciner cells of the rat exorbital lacrimal gland. J. Cell Biol. 74, , KUROSUMI, K. Cytochemical and functional morphology of the Golgi apparatus. Acta Histochem. Cytochem. 5, , LACY, D. and C.E. CHALLICE. Studies on the Golgi apparatus by electron microscopy with particular reference to Aoyama's technique. J. Biophys. Biochem. Cytol. 2, , LITTLE, J.S. and C.C. WIDNELL. Evidence for the translation of 5'-nucleotidase across hepatic membranes in vivo. Proc. Natl. Acad. Sci. U.S.A. 72, , NAGATANI, Y. and I. YAMAOKA. Studies of the Golgi substance which creates silvered Golgi apparatus in the epididymal cells of the mouse. Sci. Rep. Yamaguchi Univ. 12, 23-42, NOVIKOFF, W.B. and S. GOLDFISCHER. Nucleoside diphosphatase activity in the Golgi apparatus and its usefulness for cytological studies. Proc. Natl. Acad. Sci. U.S.A. 47, , NOVIKOFF, W.B., A.B. NOVIKOFF, N. QUINTANA and J.J. HAUW. Golgi apparatus, GERL, and lysosomes of neurons in rat dorsal root ganglion, studied by thick section and thin section cytochemistry. J. Cell Biol. 50, , SMITH, C.E. Ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase) activity to the intermediate saccules of the Golgi apparatus in rat incisor ameloblasts. J. Histochem. Cytochem. 28, 16-26, YAMAOKA, I., K. YAMAMOTO, N. URABE and Y. NAGATANI. Osmium-impregnation patterns of the Golgi complex in the epididymal epithelial cells of castrated and testosterone-injected mice. J. Cell Sci. 59, 71-79, 1983 (Received for publication, September 2, 1983)