- Email: [email protected]
Special cutaneous receptor organs of fish
1966 by Academic Press, Inc. J. U L T R A S T R U C T U R E R E S E A R C H
27, 361-372 (1969)
361
Special Cutaneous Receptor Organs of Fish V. Electroreceptor Inclusion Bodies of Eigenmannia z ALLEN W. WACHTEL AND R. BRUCE SZAMIER
Sections of Genetics and Cell Biology, and Institute of Cellular Biology, University of Connecticut, Storrs 06268 Received December 26, 1968 In receptor cells of the electroreceptors of the gymnotid fish, Eigenmannia virescens, there are inclusion bodies that contain hexagonally packed arrays of cylinders 270 ~ in diameter. The cylinders have a core of low electron density. The bodies contain RNA, acid phosphatase, and e-hydroxybutyrie acid oxidase but do not contain aryl sulfatase, catalase, or lactic acid oxidase. The bodies have not been found in receptor cells of other receptor organs or in other tissues of these fish. They appear to be unique in structure and composition.
In the course of cytological studies of electric receptors, inclusion bodies have been noted in the perinuclear cytoplasm of receptor cells in special cutaneous receptor organs of the South American weakly electric fish Eigenmannia virescens. Because of the unusual nature of some of these bodies, their structure and histochemistry are described in some detail in this report. Two types of special cutaneous receptor organs are found in great numbers on the anterior and dorsal surfaces of this fish (27, 30). The tuberous receptor organs contain many cylindrical receptor cells, supporting cells, and branches of the innervating fiber within a capsule (27, 30). A porous "plug" of specialized epithelial cells lies over the receptor cells. Ampullary receptor organs are smaller than tuberous receptor organs and contain only a few receptor cells (20, 27, 28). The duct to the surface and the extraceUular space in the capsule are filled with a jelly-like material, and epithelial plug cells are absent. Like the tuberous organs, only receptor cells, supporting cells, and branches of the nerve fiber are within the capsule. The nuclei of the receptor cells of both types of organ are always located near the distal ends of the cells. Mitochondria, elements of the Golgi apparatus, and inclusion bodies are numerous in the perinuclear cytoplasm and adjacent to the plasma Aided by a grant from The National Science Foundation. Contribution No. 169 from the Institute of Cellular Biology.
362
WACHTEL AND SZAMIER
m e m b r a n e of the distal third to half of the cell, b u t are usually absent from any other region of the cell. MATERIALS A N D METHODS Tissue for electron microscopy was prepared by the methods previously described
(28, 30). For enzymatic digestion and staining for light microscopy, segments of the fish were fixed in 4 % formaldehyde freshly prepared from paraformaldehyde, buffered with 0.07 Mphosphate at p H 7.2, at 0-5 ° for 6-24 hours. After thorough washing in water and partial dehydration in ethanol, small pieces of skin containing the desired organs were dissected from the segments. Upon completion of dehydration, the pieces of skin were embedded in diethylene glycol distearate (26), sectioned at 1-3 /z with glass knives, and mounted by drying on glass slides. Feulgen staining was done with either Basic Fuchsin or Azure A (17) 2ehiff's reagents. Methyl Green with Pyronin Y staining was done according to the procedure used by the Biological Stain Commission (6). For D N A digestion, sections were dewaxed in xylene, hydrated, and incubated for 1.5 hours at 37 ° in a 0.012 % solution of DNase (Worthington Biochemical Corp. electrophoretically purified Deoxyribonuclease I) in 0.025 M Tris and 0.0025 M MgSO~ (pH 7.2). For R N A digestion, hydrated sections were incubated for 1.5 hours at 37 o in a 1% solution of RNase (Sigma Chemical Co. Ribonuclease A, 5 x crystallized) in distilled water. For enzyme localization, segments of the fish were fixed in 4 % formaldehyde or 2.5 % glutaraldehyde, buffered as described above. Small pieces of skin were dissected free of the underlying muscle during fixation, briefly washed in the buffer used in the incubating solution, and incubated as described below. The incubating solution for acid phosphatase localization contained 0.1% sodium ~naphthyl phosphate and 0.08 % hexazonium pararosanilin (8) in 0.07 M phosphate buffer at pH 6.4. Incubation was carried out at 0-5 ° for varying times. Controls were incubated without substrate. After incubation the tissue was embedded in diethylene glycol distearate. FIG. 1. Receptor cells of a tuberous receptor organ of Eigenmannia. Several inclusion bodies (arrows) in the perinuclear cytoplasm are distinguished from the surrounding mitochondria by their greater size or density. The dense oval bodies at the bases of the receptor cells are nerve endings which are crowded with many mitochondria. Cuboidal supporting cells are below the nerve endings. Epithelial "plug" cells lie above the receptor cells. N, nucleus; P, plug; S, supporting cell. Toluidine blue stained Epon section, x 2700. FIG. 2. Parts of two adjacent tuberous receptor organs incubated for acid phosphatase localization. Dense reaction product is seen in the inclusion bodies in the receptor cells. A few small granules in the supporting cells (S) also were active. The dense object at the top is a necrotic plug cell. N, nucleus. x 1800. FIG. 3. Nonenzymatic staining of tuberous receptor organ incubated with diaminobenzidine and hydrogen peroxide. Mitochondria in the receptor cells, the nerve endings, and the nerve fiber (dense objects at bottom edge) stained selectively. Dense object at top is a necrotic plug cell that also stained. Dense object at left is a pigment cell. Inclusion bodies did not stain, x 950. Fro. 4. Distal third of receptor cell of tuberous receptor organ. The largest inclusion body (arrow at right) contains several dense bundles of cylinders. Each of the smaller inclusion bodies (arrows at left) to the left of the nuclear cap (N) contains only one bundle of cylinders. At higher magnification more cylinders could be seen in these inclusion bodies. The space between adjacent receptor cells and between the receptor cells and the plug cells (P) is filled with the intracapsular fluid. Compare with Fig. 1. x 9600.
!ii~ii!ii!iiii~i!i~!!~i~ii~ij~!! ¸
~i~
364
WACHTEL AND SZAMIER
Aryl sulfatase localization was done according to the method of Goldfischer (14). Fixed pieces of skin were incubated at 0 ° or at 37 ° withp-nitrocatechol sulfate (Sigma Chemical Co.) in the presence of lead nitrate in Veronal acetate buffer at pH 4.2 and 5.5. After incubation the tissue was washed in buffer, treated with dilute ammonium sulfide, then embedded in diethylene glycol distearate. Peroxidase localization was attempted using the incubating solution described by Graham and Karnovsky (15). Fixed skin was incubated at 0 ° or at 37 ° in solutions containing 3,3'diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) and hydrogen peroxide in Tris buffer at pH 7.6. After incubation the tissues were treated with potassium ferricyanide solution and embedded in diethylene glycol distearate, or postfixed in 2 % OsO4 and embedded in DER 334 (34) for electron microscopy. For u-hydroxy acid oxidase localization, tissue was treated by the method of Allen and Beard (1). Fixed skin was incubated at pH 7.5 with either og-lactate or De-~-hydroxybutyrate. In each case the incubation solution contained phenazine methosulfate and the tetrazolium salt Nitro BT. After incubation, tissues were embedded in diethylene glycol distearate or postfixed and embedded in DER 334.
RESULTS AND OBSERVATIONS
Light microscopy and histochemistry In almost every tuberous receptor organ that we have examined, inclusion bodies are seen in every thick section that passes through the nuclei of the receptor cells. In thin sections they are not always found, but this is accounted for by geometric considerations. They seem to be ubiquitous components of the receptor cells of the tuberous receptor organs, and usually they are present in ampullary receptor organs. In 1- to 2-g sections of osmium-fixed, epoxy or polyester-embedded receptor organs, they are seen as dense bodies among the mitochondria in the perinuclear cytoplasm of receptor cells of both types of special cutaneous receptor organs (Fig. 1). They are not present in any other regions of the cells. In toluidine blue-stained sections viewed by transmitted light, or unstained sections viewed by phase contrast microscopy, the nucleoli and the inclusion bodies appear equally dense and are the densest structures within these cells. The smaller inclusion bodies are readily distinguished by their greater density from mitochondria of similar size. The largest inclusion bodies are as much as 2 # in diameter and are not easily confused with other cell structures. Feulgen staining of sections of aldehyde-fixed, diethylene glycol distearate-embedded tissue with either of the Schiff's reagents following HC1 hydrolysis results in the expected nuclear staining pattern, but does not color the cytoplasmic bodies. Methyl FIGS. 5-7. Each inclusion body contains one or several bundles of cylinders with regular hexagonal packing, as well as some single cylinders, granules, and small vesicles. The longest cylinders in Fig. 6 are almost 1/~ tong. Note single membrane surrounding inclusion bodies. Fig. 5, x 41,000; Fig. 6, × 45,500; Fig. 7, x 66,000.
24
691831 J . Ultrastructure ReseaTch
366
WACHTEL AND SZAMIER
Green-Pyronin Y staining produces a strong rose color in the inclusion bodies, comparable to the reaction in the nucleoli. Adjacent sections were treated with the nuclease solutions described. After DNase treatment, Pyronin staining of the inclusion bodies was unchanged. After RNase treatment, Pyronin staining of the inclusion bodies as well as of the nucleoli was not detectable. Long incubations (6 hours to overnight) in the complete acid phosphate medium resulted in the deposit of dense red precipitate in the inclusion bodies in the receptor ceils and i n a few granules in the supporting cells and some plug cells of tuberous organs (Fig. 2). Reaction product was not visible in adjacent tissues in the sections and was entirely absent from tissue incubated without substrate. Similar localization could be demonstrated after even 6 days in formalin. Short incubation (ca. l - 2 hours) produced no visible reaction product even in briefly fixed (ca. 1-2 hours) tissue. After incubation with p-nitrocatechol sulfate at either p H 4.2 or 5.5 scattered reaction product was observed in receptor cells, supporting ceils, and in plug cells of tuberous organs. The reaction was slower at 0 ° than at 37 ° but was otherwise the same. The reaction product was not associated with the inclusion bodies that could be seen in the same sections by phase contrast microscopy. In tissues incubated without substrate, a few lead crystals were present on the surface of the tissue, but were not observed within the cells. Incubation with diaminobenzidine produced a visible color reaction in the receptor organs within one-half hour at 37 °. The color deepened with time, and was more intense if hydrogen peroxide were present. Hydrogen peroxide was not necessary for the reaction. After overnight fixation the color developed more slowly; however, development was accelerated by heating the tissue to 70 ° . In each case, microscopic examination showed that the color was localized in the mitochondria of the receptor cells and the nerve endings (Fig. 3). In the supporting cells, and the surrounding epidermis and connective tissue, in which there are fewer mitochondria, fewer colored structures were noted. In the electron microscope the restriction to mitochondria was confirmed. Electron dense product was present between the inner and outer membranes and within the cristae. No reaction was observed in inclusion bodies. Results of incubations for ~-hydroxy acid oxidase localization were completely negative in both light and electron microscope preparations when lactate was used as a substrate. With ~-hydroxybutyrate present in the incubation mixture, inclusion bodies were slightly colored. Other cell structures were not colored by either of these FIG. 8. The regular packing of the cylinders is evident in bundles that are cut obliquely or in cross section. The bundle at the upper right is seen at higher magnification in Fig. 9. x 57,000.
8
;
368
W A C H T E L A N D SZAMIER
incubation procedures, and the inclusion bodies were not colored by incubation in control solutions from which the substrate had been omitted.
Electron microscopy In electron micrographs of plastic-embedded osmium-fixed receptor cells, inclusion bodies are found that correspond in size and distribution to the toluidine bluestainable dense bodies seen in light microscope preparations (Fig. 4). These range in diameter from 2/~, which correspond to the largest dense bodies discernible in the light microscope, to about 0.2/~. All are limited by a single membrane (Figs. 5-7). Some contain on!y randomly distributed granular or vesicular material. The great majority also contain dense crystaMike aggregates of cylindrical structures (Figs. 5-8). The cylinders have a uniform diameter of about 270 A, and a core of low electron density about 100 A in diameter. There is some indication that subunits form the walls of the cylinders (Fig. 9), but rotation analysis by Markham's method (22) with n ~<16 has not revealed any reproducible number of substructures. The cylinders are closely packed in bundles with hexagonal order (Figs. 7-9). Some inclusion bodies are almost filled by one or several bundles, but in most they occupy a relatively small part of the membrane-contained volume. Commonly several bundles of cylinders in different planes as well as some free cylinders occupy an inclusion body. The lengths of the cylinders vary. The lengths of 100 that appeared to lie entirely within the plane of the sections were measured. The mean length was 0.48 #, and the longest was 0.80/~ (Fig. 6). The distribution of lengths approximates a normal curve. Cylinders have never been observed in these fish except within the cytoplasmic inclusion bodies in the receptor cells of both types of special cutaneous receptor organs. Free cylinders have not been observed in the cells, extracellular fluid, or jelly. A survey of other cell types and tissues of these fish by both light and electron microscopy has not yet revealed similar inclusion bodies or cylinders in any of them. These include the supporting cells, capsule cells and nerve fibers that are in intimate contact with the receptor cells of the special cutaneous receptor organs, as well as the skin, blood, corium, kidney, liver, brain, and receptor cells of free neuromasts and lateral line canal receptor organs. Cytoplasmic microtubules are present in accessory cells and in receptor cells. Though of similar diameter to the cylinders of inclusion bodies, they differ morphologically from the cylinders in having greater length, thinner walls, and lower electron density. They are not membrane enclosed and do not form ordered arrays even when several are present in close proximity to each other.
ELECTRORECEPTOR INCLUSION BODIES
369
FIG. 9. In cross sections viewed at high magnification there sometimes appears to be an indication of substructures. However, no regular periodicity has been revealed by rotation analysis, x 450,000.
370
W A C H T E L AND SZAMIER
DISCUSSION Inclusion bodies of many sorts have been described in various animal cells. A few types have been reported in fish receptor cells. The receptor cells of the small pit organs of Amiurus contain large crystalline inclusions which are not enclosed in membranes and also some vesicles 0.4-0.8 # in diameter that contain patches of dense noncrystalline material (23). Receptor cells in minnow epidermis and fish taste bud.s contain small dense inclusions (29, 33). None of these are structurally like the inclusion bodies that we have described here. Ordinary lateral line canal organ receptor ceils do not have similar inclusions in either the Eigenmannia that we have studied or in other species (12, 13, 16, 25). They have not been reported in receptor cells of the special cutaneous receptor organs of other electric fish (2, 3, 10, 27, 32), although we have observed them in the tuberous organs of one other South American gymnotid genus closely related to Eigenmannia (unpublished observations). They are not present in the ampullary organs of the nonelectric catfish Kryptopterus (31). In another study of the fine structure of the special cutaneous receptor organs of Eigenmannia, inclusion bodies were found in the receptor cells of the tuberous organs, but the cylinders were not observed (20). The bodies may be one of several kinds that have been observed in animal cells: lysosomes, microbodies, or virus inclusion bodies. The presence of the cylinders, the restriction of these bodies to a single cell type, the high R N A content, and the absence of aryl sulfatase activity from the bodies, though it does appear to be present in the receptor cells, would all tend to indicate that these bodies are not lysosomes. However, they do contain one of the more characteristic lysosomal enzymes, acid phosphatase. The possibility that the inclusion bodies are microbodies is considered because of the presence of the cylinders. Microbodies of rat liver contain dense cores made up of hexagonally packed tubules up to 115 & in diameter (18). Such tubules possibly consist of urate oxidase or catalase which are present in microbodies in high concentration (4, 9, 21). Purified catalase has been shown to crystallize under some conditions as tubules with diameters of 310 & or 420 A, depending on the source of the protein (19). In these tubular crystals, the walls are monomolecular, 65 A in thickness, and formed of two step helices with 23 molecules per turn. Subunits in a wall of such structure would not be easily detected by rotation analysis. Under similar conditions to those that we employed for catalase localization, Novikoff and Goldfisher (24) and Fahimi (11) found that rat liver microbodies were reactive. The nonenzymatic mitochondrial reaction that we found was also found in some cell types by Novikoff and Goldfisher (24). ~-Hydroxy acid oxidase is another enzyme that has been associated with microbodies both biochemically (1, 4) and histochemically (1).
ELECTRORECEPTOR INCLUSION BODIES
371
Both lactate and ~-hydroxybutyrate are oxidized by the microbody enzymes (9). The substrate specificity of the inclusion body oxidase, and the absence of catalase activity indicate that the inclusion bodies in Eigenmannia are not microbodies. If the cylinders are tubular crystals of a protein that is present in high concentration, it is some protein other than catalase. The possibility that the bodies are virus inclusion bodies is considered for several reasons. The receptor cells are not deciduous and are relatively readily accessible to external agents. The cylinders of Eigenmannia inclusion bodies (4800 A in length and 270 A in diameter) resemble some of the elongated plant viruses such as those in G r o u p 3 of Brandes and Wetter's (5) classification that includes T M V (3000 A in length and 150 A in diameter), and the possibly viral rhapidosomes of Saprospira (2250 ~ in length and 330 A in diameter) (7) in structure and R N A content. Still, we have not yet been able to isolate cylinders for analysis, electron microscope studies of aldehyde-fixed ribonuclease-digested organs have not enabled us to determine where in the inclusion bodies the nucleic acid is localized, and elongate R N A viruses have not been found in animal cells so far as we are aware. The restriction of these inclusion bodies to the receptor cells of the special cutaneous receptor organs of gymnotids and the unique structure of the bodies probably indicate that they are related to the specific functional activities of the receptors, although such a functional relationship cannot be defined at this time. If this is the case, they need not necessarily closely resemble any of the bodies with which we have compared them but might have unique composition as well as structure.
REFERENCES 1. ALLEN, J, and BEARD, M., Science 149, 1507 (1965). 2. BARETS,A. and SZABO, T., J. Microscopie 1, 47 (1962). 3. - ibid. 3, 85 (1964). 4. BAUDHUIN,P., BEAUFAY,H. and DE DUVE, C., J. Cell BioL 26, 219 (1965). 5. BRANDES,J. and WETTER, C., Virology 8, 99 (1959). 6. CONN, H. J., DARROW, M. A. and EMMEL,V. M., Staining Procedures Used by the Biological Stain Commission, pp, 166-167. Williams & Wilkins, Baltimore, Maryland, 1960. 7. CORRELL,D. L. and LEWIN, R. A., Can. Y. Microbiol. 10, 63 (1964). 8. DAVIS, B. J., Proc. Soc. Exptl. Biol. Med. 101, 90 (1959). 9. DE DUVE, C. and BAUDHUIN,P., Physiol. Rev. 46, 323 (1966). 10. DERBIN, C. and SZABO, T., Y. Ultrastructure Res. 22, 469 (1968). 11. FAHIMI,H. D., 9". Histochem. Cytochem. 16, 547 (1968). 12. FLOCK, ~., Acta Laryngol. 199, 7 (1965). 13. - Lateral Line Detectors, p. 163. Indiana Univ. Press, Bloomington, Indiana, 1967. 14. GOLDF~SHER,S., J. Histochem. Cytochem. 13, 520 (1965).
372 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
WACHTELAND SZAMIER
GRAHAM,R. C., JR. and KARNOVSKY,M. J., J. Histochem. Cytochem. 14, 291 (1966). HAMA,K., J. Cell Biol. 24, 193 (1965). HIMES,M. and MORmER, L., Stain Technol. 31, 67 (1956). HRUBAN,Z. and SwIFa', H., Science 146, 1316 (1964). KISLEV,N. A., SHPITZBERG,C. L. and VAINSHTEIN,B. K., J. Mol. Biol. 25, 433 (1967). LISSMANN,H. W. and MULLINGER,A. M., Proc. Roy. Soe. B169, 345 (1968). MAHLER,H. R., HOBSCHER,G. and BAUM, H., J. Biol. Chem. 216, 625 (1955). MARKHAM,R., Virology 20, 88 (1963). MULLINGER,A. M., Proc. Roy. Soe. B160, 345 (1964). NOVIKOFF,A. B. and GOLDFISHER,S., J. Histoehem. Cytoehem. 16, 507 (1968). PETRAIT~S,R., J. Morphol. 118, 367 (1966). SALAZAR,H., Stain Technol. 39, 13 (1964). SZABO,T., J. Morphol. 117, 229 (1965). SZAMIER,R. B. and WACHTEL,A., Y. Morphol. in press (1969). TRtSJILLo-CEN6Z,0., Z. Zellforseh. Mikroskop. Anat. 54, 654 (1961). WACHTEL,A. and SZAMmR,R. B., or. Morphol. 119, 51 (1966). - - - - ibid. in press (1969). 32. WALTMAN,B., Aeta Physiol. Seand. 66, Suppl. 264, 1 (1966). 33. WHXTEAR,M., Nature 208, 703 (1965). 34. WINBORN,W., Stain Teehnol. 40, 227 (1965).
27, 361-372 (1969)
361
Special Cutaneous Receptor Organs of Fish V. Electroreceptor Inclusion Bodies of Eigenmannia z ALLEN W. WACHTEL AND R. BRUCE SZAMIER
Sections of Genetics and Cell Biology, and Institute of Cellular Biology, University of Connecticut, Storrs 06268 Received December 26, 1968 In receptor cells of the electroreceptors of the gymnotid fish, Eigenmannia virescens, there are inclusion bodies that contain hexagonally packed arrays of cylinders 270 ~ in diameter. The cylinders have a core of low electron density. The bodies contain RNA, acid phosphatase, and e-hydroxybutyrie acid oxidase but do not contain aryl sulfatase, catalase, or lactic acid oxidase. The bodies have not been found in receptor cells of other receptor organs or in other tissues of these fish. They appear to be unique in structure and composition.
In the course of cytological studies of electric receptors, inclusion bodies have been noted in the perinuclear cytoplasm of receptor cells in special cutaneous receptor organs of the South American weakly electric fish Eigenmannia virescens. Because of the unusual nature of some of these bodies, their structure and histochemistry are described in some detail in this report. Two types of special cutaneous receptor organs are found in great numbers on the anterior and dorsal surfaces of this fish (27, 30). The tuberous receptor organs contain many cylindrical receptor cells, supporting cells, and branches of the innervating fiber within a capsule (27, 30). A porous "plug" of specialized epithelial cells lies over the receptor cells. Ampullary receptor organs are smaller than tuberous receptor organs and contain only a few receptor cells (20, 27, 28). The duct to the surface and the extraceUular space in the capsule are filled with a jelly-like material, and epithelial plug cells are absent. Like the tuberous organs, only receptor cells, supporting cells, and branches of the nerve fiber are within the capsule. The nuclei of the receptor cells of both types of organ are always located near the distal ends of the cells. Mitochondria, elements of the Golgi apparatus, and inclusion bodies are numerous in the perinuclear cytoplasm and adjacent to the plasma Aided by a grant from The National Science Foundation. Contribution No. 169 from the Institute of Cellular Biology.
362
WACHTEL AND SZAMIER
m e m b r a n e of the distal third to half of the cell, b u t are usually absent from any other region of the cell. MATERIALS A N D METHODS Tissue for electron microscopy was prepared by the methods previously described
(28, 30). For enzymatic digestion and staining for light microscopy, segments of the fish were fixed in 4 % formaldehyde freshly prepared from paraformaldehyde, buffered with 0.07 Mphosphate at p H 7.2, at 0-5 ° for 6-24 hours. After thorough washing in water and partial dehydration in ethanol, small pieces of skin containing the desired organs were dissected from the segments. Upon completion of dehydration, the pieces of skin were embedded in diethylene glycol distearate (26), sectioned at 1-3 /z with glass knives, and mounted by drying on glass slides. Feulgen staining was done with either Basic Fuchsin or Azure A (17) 2ehiff's reagents. Methyl Green with Pyronin Y staining was done according to the procedure used by the Biological Stain Commission (6). For D N A digestion, sections were dewaxed in xylene, hydrated, and incubated for 1.5 hours at 37 ° in a 0.012 % solution of DNase (Worthington Biochemical Corp. electrophoretically purified Deoxyribonuclease I) in 0.025 M Tris and 0.0025 M MgSO~ (pH 7.2). For R N A digestion, hydrated sections were incubated for 1.5 hours at 37 o in a 1% solution of RNase (Sigma Chemical Co. Ribonuclease A, 5 x crystallized) in distilled water. For enzyme localization, segments of the fish were fixed in 4 % formaldehyde or 2.5 % glutaraldehyde, buffered as described above. Small pieces of skin were dissected free of the underlying muscle during fixation, briefly washed in the buffer used in the incubating solution, and incubated as described below. The incubating solution for acid phosphatase localization contained 0.1% sodium ~naphthyl phosphate and 0.08 % hexazonium pararosanilin (8) in 0.07 M phosphate buffer at pH 6.4. Incubation was carried out at 0-5 ° for varying times. Controls were incubated without substrate. After incubation the tissue was embedded in diethylene glycol distearate. FIG. 1. Receptor cells of a tuberous receptor organ of Eigenmannia. Several inclusion bodies (arrows) in the perinuclear cytoplasm are distinguished from the surrounding mitochondria by their greater size or density. The dense oval bodies at the bases of the receptor cells are nerve endings which are crowded with many mitochondria. Cuboidal supporting cells are below the nerve endings. Epithelial "plug" cells lie above the receptor cells. N, nucleus; P, plug; S, supporting cell. Toluidine blue stained Epon section, x 2700. FIG. 2. Parts of two adjacent tuberous receptor organs incubated for acid phosphatase localization. Dense reaction product is seen in the inclusion bodies in the receptor cells. A few small granules in the supporting cells (S) also were active. The dense object at the top is a necrotic plug cell. N, nucleus. x 1800. FIG. 3. Nonenzymatic staining of tuberous receptor organ incubated with diaminobenzidine and hydrogen peroxide. Mitochondria in the receptor cells, the nerve endings, and the nerve fiber (dense objects at bottom edge) stained selectively. Dense object at top is a necrotic plug cell that also stained. Dense object at left is a pigment cell. Inclusion bodies did not stain, x 950. Fro. 4. Distal third of receptor cell of tuberous receptor organ. The largest inclusion body (arrow at right) contains several dense bundles of cylinders. Each of the smaller inclusion bodies (arrows at left) to the left of the nuclear cap (N) contains only one bundle of cylinders. At higher magnification more cylinders could be seen in these inclusion bodies. The space between adjacent receptor cells and between the receptor cells and the plug cells (P) is filled with the intracapsular fluid. Compare with Fig. 1. x 9600.
!ii~ii!ii!iiii~i!i~!!~i~ii~ij~!! ¸
~i~
364
WACHTEL AND SZAMIER
Aryl sulfatase localization was done according to the method of Goldfischer (14). Fixed pieces of skin were incubated at 0 ° or at 37 ° withp-nitrocatechol sulfate (Sigma Chemical Co.) in the presence of lead nitrate in Veronal acetate buffer at pH 4.2 and 5.5. After incubation the tissue was washed in buffer, treated with dilute ammonium sulfide, then embedded in diethylene glycol distearate. Peroxidase localization was attempted using the incubating solution described by Graham and Karnovsky (15). Fixed skin was incubated at 0 ° or at 37 ° in solutions containing 3,3'diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) and hydrogen peroxide in Tris buffer at pH 7.6. After incubation the tissues were treated with potassium ferricyanide solution and embedded in diethylene glycol distearate, or postfixed in 2 % OsO4 and embedded in DER 334 (34) for electron microscopy. For u-hydroxy acid oxidase localization, tissue was treated by the method of Allen and Beard (1). Fixed skin was incubated at pH 7.5 with either og-lactate or De-~-hydroxybutyrate. In each case the incubation solution contained phenazine methosulfate and the tetrazolium salt Nitro BT. After incubation, tissues were embedded in diethylene glycol distearate or postfixed and embedded in DER 334.
RESULTS AND OBSERVATIONS
Light microscopy and histochemistry In almost every tuberous receptor organ that we have examined, inclusion bodies are seen in every thick section that passes through the nuclei of the receptor cells. In thin sections they are not always found, but this is accounted for by geometric considerations. They seem to be ubiquitous components of the receptor cells of the tuberous receptor organs, and usually they are present in ampullary receptor organs. In 1- to 2-g sections of osmium-fixed, epoxy or polyester-embedded receptor organs, they are seen as dense bodies among the mitochondria in the perinuclear cytoplasm of receptor cells of both types of special cutaneous receptor organs (Fig. 1). They are not present in any other regions of the cells. In toluidine blue-stained sections viewed by transmitted light, or unstained sections viewed by phase contrast microscopy, the nucleoli and the inclusion bodies appear equally dense and are the densest structures within these cells. The smaller inclusion bodies are readily distinguished by their greater density from mitochondria of similar size. The largest inclusion bodies are as much as 2 # in diameter and are not easily confused with other cell structures. Feulgen staining of sections of aldehyde-fixed, diethylene glycol distearate-embedded tissue with either of the Schiff's reagents following HC1 hydrolysis results in the expected nuclear staining pattern, but does not color the cytoplasmic bodies. Methyl FIGS. 5-7. Each inclusion body contains one or several bundles of cylinders with regular hexagonal packing, as well as some single cylinders, granules, and small vesicles. The longest cylinders in Fig. 6 are almost 1/~ tong. Note single membrane surrounding inclusion bodies. Fig. 5, x 41,000; Fig. 6, × 45,500; Fig. 7, x 66,000.
24
691831 J . Ultrastructure ReseaTch
366
WACHTEL AND SZAMIER
Green-Pyronin Y staining produces a strong rose color in the inclusion bodies, comparable to the reaction in the nucleoli. Adjacent sections were treated with the nuclease solutions described. After DNase treatment, Pyronin staining of the inclusion bodies was unchanged. After RNase treatment, Pyronin staining of the inclusion bodies as well as of the nucleoli was not detectable. Long incubations (6 hours to overnight) in the complete acid phosphate medium resulted in the deposit of dense red precipitate in the inclusion bodies in the receptor ceils and i n a few granules in the supporting cells and some plug cells of tuberous organs (Fig. 2). Reaction product was not visible in adjacent tissues in the sections and was entirely absent from tissue incubated without substrate. Similar localization could be demonstrated after even 6 days in formalin. Short incubation (ca. l - 2 hours) produced no visible reaction product even in briefly fixed (ca. 1-2 hours) tissue. After incubation with p-nitrocatechol sulfate at either p H 4.2 or 5.5 scattered reaction product was observed in receptor cells, supporting ceils, and in plug cells of tuberous organs. The reaction was slower at 0 ° than at 37 ° but was otherwise the same. The reaction product was not associated with the inclusion bodies that could be seen in the same sections by phase contrast microscopy. In tissues incubated without substrate, a few lead crystals were present on the surface of the tissue, but were not observed within the cells. Incubation with diaminobenzidine produced a visible color reaction in the receptor organs within one-half hour at 37 °. The color deepened with time, and was more intense if hydrogen peroxide were present. Hydrogen peroxide was not necessary for the reaction. After overnight fixation the color developed more slowly; however, development was accelerated by heating the tissue to 70 ° . In each case, microscopic examination showed that the color was localized in the mitochondria of the receptor cells and the nerve endings (Fig. 3). In the supporting cells, and the surrounding epidermis and connective tissue, in which there are fewer mitochondria, fewer colored structures were noted. In the electron microscope the restriction to mitochondria was confirmed. Electron dense product was present between the inner and outer membranes and within the cristae. No reaction was observed in inclusion bodies. Results of incubations for ~-hydroxy acid oxidase localization were completely negative in both light and electron microscope preparations when lactate was used as a substrate. With ~-hydroxybutyrate present in the incubation mixture, inclusion bodies were slightly colored. Other cell structures were not colored by either of these FIG. 8. The regular packing of the cylinders is evident in bundles that are cut obliquely or in cross section. The bundle at the upper right is seen at higher magnification in Fig. 9. x 57,000.
8
;
368
W A C H T E L A N D SZAMIER
incubation procedures, and the inclusion bodies were not colored by incubation in control solutions from which the substrate had been omitted.
Electron microscopy In electron micrographs of plastic-embedded osmium-fixed receptor cells, inclusion bodies are found that correspond in size and distribution to the toluidine bluestainable dense bodies seen in light microscope preparations (Fig. 4). These range in diameter from 2/~, which correspond to the largest dense bodies discernible in the light microscope, to about 0.2/~. All are limited by a single membrane (Figs. 5-7). Some contain on!y randomly distributed granular or vesicular material. The great majority also contain dense crystaMike aggregates of cylindrical structures (Figs. 5-8). The cylinders have a uniform diameter of about 270 A, and a core of low electron density about 100 A in diameter. There is some indication that subunits form the walls of the cylinders (Fig. 9), but rotation analysis by Markham's method (22) with n ~<16 has not revealed any reproducible number of substructures. The cylinders are closely packed in bundles with hexagonal order (Figs. 7-9). Some inclusion bodies are almost filled by one or several bundles, but in most they occupy a relatively small part of the membrane-contained volume. Commonly several bundles of cylinders in different planes as well as some free cylinders occupy an inclusion body. The lengths of the cylinders vary. The lengths of 100 that appeared to lie entirely within the plane of the sections were measured. The mean length was 0.48 #, and the longest was 0.80/~ (Fig. 6). The distribution of lengths approximates a normal curve. Cylinders have never been observed in these fish except within the cytoplasmic inclusion bodies in the receptor cells of both types of special cutaneous receptor organs. Free cylinders have not been observed in the cells, extracellular fluid, or jelly. A survey of other cell types and tissues of these fish by both light and electron microscopy has not yet revealed similar inclusion bodies or cylinders in any of them. These include the supporting cells, capsule cells and nerve fibers that are in intimate contact with the receptor cells of the special cutaneous receptor organs, as well as the skin, blood, corium, kidney, liver, brain, and receptor cells of free neuromasts and lateral line canal receptor organs. Cytoplasmic microtubules are present in accessory cells and in receptor cells. Though of similar diameter to the cylinders of inclusion bodies, they differ morphologically from the cylinders in having greater length, thinner walls, and lower electron density. They are not membrane enclosed and do not form ordered arrays even when several are present in close proximity to each other.
ELECTRORECEPTOR INCLUSION BODIES
369
FIG. 9. In cross sections viewed at high magnification there sometimes appears to be an indication of substructures. However, no regular periodicity has been revealed by rotation analysis, x 450,000.
370
W A C H T E L AND SZAMIER
DISCUSSION Inclusion bodies of many sorts have been described in various animal cells. A few types have been reported in fish receptor cells. The receptor cells of the small pit organs of Amiurus contain large crystalline inclusions which are not enclosed in membranes and also some vesicles 0.4-0.8 # in diameter that contain patches of dense noncrystalline material (23). Receptor cells in minnow epidermis and fish taste bud.s contain small dense inclusions (29, 33). None of these are structurally like the inclusion bodies that we have described here. Ordinary lateral line canal organ receptor ceils do not have similar inclusions in either the Eigenmannia that we have studied or in other species (12, 13, 16, 25). They have not been reported in receptor cells of the special cutaneous receptor organs of other electric fish (2, 3, 10, 27, 32), although we have observed them in the tuberous organs of one other South American gymnotid genus closely related to Eigenmannia (unpublished observations). They are not present in the ampullary organs of the nonelectric catfish Kryptopterus (31). In another study of the fine structure of the special cutaneous receptor organs of Eigenmannia, inclusion bodies were found in the receptor cells of the tuberous organs, but the cylinders were not observed (20). The bodies may be one of several kinds that have been observed in animal cells: lysosomes, microbodies, or virus inclusion bodies. The presence of the cylinders, the restriction of these bodies to a single cell type, the high R N A content, and the absence of aryl sulfatase activity from the bodies, though it does appear to be present in the receptor cells, would all tend to indicate that these bodies are not lysosomes. However, they do contain one of the more characteristic lysosomal enzymes, acid phosphatase. The possibility that the inclusion bodies are microbodies is considered because of the presence of the cylinders. Microbodies of rat liver contain dense cores made up of hexagonally packed tubules up to 115 & in diameter (18). Such tubules possibly consist of urate oxidase or catalase which are present in microbodies in high concentration (4, 9, 21). Purified catalase has been shown to crystallize under some conditions as tubules with diameters of 310 & or 420 A, depending on the source of the protein (19). In these tubular crystals, the walls are monomolecular, 65 A in thickness, and formed of two step helices with 23 molecules per turn. Subunits in a wall of such structure would not be easily detected by rotation analysis. Under similar conditions to those that we employed for catalase localization, Novikoff and Goldfisher (24) and Fahimi (11) found that rat liver microbodies were reactive. The nonenzymatic mitochondrial reaction that we found was also found in some cell types by Novikoff and Goldfisher (24). ~-Hydroxy acid oxidase is another enzyme that has been associated with microbodies both biochemically (1, 4) and histochemically (1).
ELECTRORECEPTOR INCLUSION BODIES
371
Both lactate and ~-hydroxybutyrate are oxidized by the microbody enzymes (9). The substrate specificity of the inclusion body oxidase, and the absence of catalase activity indicate that the inclusion bodies in Eigenmannia are not microbodies. If the cylinders are tubular crystals of a protein that is present in high concentration, it is some protein other than catalase. The possibility that the bodies are virus inclusion bodies is considered for several reasons. The receptor cells are not deciduous and are relatively readily accessible to external agents. The cylinders of Eigenmannia inclusion bodies (4800 A in length and 270 A in diameter) resemble some of the elongated plant viruses such as those in G r o u p 3 of Brandes and Wetter's (5) classification that includes T M V (3000 A in length and 150 A in diameter), and the possibly viral rhapidosomes of Saprospira (2250 ~ in length and 330 A in diameter) (7) in structure and R N A content. Still, we have not yet been able to isolate cylinders for analysis, electron microscope studies of aldehyde-fixed ribonuclease-digested organs have not enabled us to determine where in the inclusion bodies the nucleic acid is localized, and elongate R N A viruses have not been found in animal cells so far as we are aware. The restriction of these inclusion bodies to the receptor cells of the special cutaneous receptor organs of gymnotids and the unique structure of the bodies probably indicate that they are related to the specific functional activities of the receptors, although such a functional relationship cannot be defined at this time. If this is the case, they need not necessarily closely resemble any of the bodies with which we have compared them but might have unique composition as well as structure.
REFERENCES 1. ALLEN, J, and BEARD, M., Science 149, 1507 (1965). 2. BARETS,A. and SZABO, T., J. Microscopie 1, 47 (1962). 3. - ibid. 3, 85 (1964). 4. BAUDHUIN,P., BEAUFAY,H. and DE DUVE, C., J. Cell BioL 26, 219 (1965). 5. BRANDES,J. and WETTER, C., Virology 8, 99 (1959). 6. CONN, H. J., DARROW, M. A. and EMMEL,V. M., Staining Procedures Used by the Biological Stain Commission, pp, 166-167. Williams & Wilkins, Baltimore, Maryland, 1960. 7. CORRELL,D. L. and LEWIN, R. A., Can. Y. Microbiol. 10, 63 (1964). 8. DAVIS, B. J., Proc. Soc. Exptl. Biol. Med. 101, 90 (1959). 9. DE DUVE, C. and BAUDHUIN,P., Physiol. Rev. 46, 323 (1966). 10. DERBIN, C. and SZABO, T., Y. Ultrastructure Res. 22, 469 (1968). 11. FAHIMI,H. D., 9". Histochem. Cytochem. 16, 547 (1968). 12. FLOCK, ~., Acta Laryngol. 199, 7 (1965). 13. - Lateral Line Detectors, p. 163. Indiana Univ. Press, Bloomington, Indiana, 1967. 14. GOLDF~SHER,S., J. Histochem. Cytochem. 13, 520 (1965).
372 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
WACHTELAND SZAMIER
GRAHAM,R. C., JR. and KARNOVSKY,M. J., J. Histochem. Cytochem. 14, 291 (1966). HAMA,K., J. Cell Biol. 24, 193 (1965). HIMES,M. and MORmER, L., Stain Technol. 31, 67 (1956). HRUBAN,Z. and SwIFa', H., Science 146, 1316 (1964). KISLEV,N. A., SHPITZBERG,C. L. and VAINSHTEIN,B. K., J. Mol. Biol. 25, 433 (1967). LISSMANN,H. W. and MULLINGER,A. M., Proc. Roy. Soe. B169, 345 (1968). MAHLER,H. R., HOBSCHER,G. and BAUM, H., J. Biol. Chem. 216, 625 (1955). MARKHAM,R., Virology 20, 88 (1963). MULLINGER,A. M., Proc. Roy. Soe. B160, 345 (1964). NOVIKOFF,A. B. and GOLDFISHER,S., J. Histoehem. Cytoehem. 16, 507 (1968). PETRAIT~S,R., J. Morphol. 118, 367 (1966). SALAZAR,H., Stain Technol. 39, 13 (1964). SZABO,T., J. Morphol. 117, 229 (1965). SZAMIER,R. B. and WACHTEL,A., Y. Morphol. in press (1969). TRtSJILLo-CEN6Z,0., Z. Zellforseh. Mikroskop. Anat. 54, 654 (1961). WACHTEL,A. and SZAMmR,R. B., or. Morphol. 119, 51 (1966). - - - - ibid. in press (1969). 32. WALTMAN,B., Aeta Physiol. Seand. 66, Suppl. 264, 1 (1966). 33. WHXTEAR,M., Nature 208, 703 (1965). 34. WINBORN,W., Stain Teehnol. 40, 227 (1965).