Albrecht v. Graefes Arch. klin. exp. Ophthal. 2Ol, 1-1o (1976)
Graefes Archiv for klinischeundexperimentelle
Ophthalmologie ,~ by Springer-Verlag 1976
Cryoultramicroscopy of Lower Vertebrate Retina Nobuo Maekawa and Katsuyoshi Mizuno Department of Ophthalmology, Tohoku University School of Medicine, Seiryo-cho, Sendai, 980 Japan Summary. Cryoultramicroscopy was carried out in the retina of lower vertebrates. Myeloid bodies of the pigment epithelium of turtles and synaptic vesicles and ribbons of frogs were stained negatively. Numerous altered, shaped, dense bodies, containing granular and myelinlike matrices, were found in the retinal pigment epithelium of turtle, and were thought to be lysosome. The advantages and disadvantages of this method are discussed.
Introduction Since the first report of cryoultramicroscopy by Fernandez-Moran and Orville (1952) had been presented, several authors (Koda, 1973, 1974 a, b; Koda and Mizuno, 1974) attempted to observe the retina by cryoultramicroscopy, but without satisfactory results. Thus, study of the fine structure of the retina with this technique was a difficult matter, because of its technical problems to preserve the ultrathin frozen section. The difficulties were partly overcome with the new technique, which improves preservation of the ultrathin frozen sections and improves the pick-up of sections by using sucrose (Tokuyasu, 1973). This new technique seems worthwhile from the principle and practice of cryoultramicroscopy. In our previous report (Maekawa and Mizuno, 1976), the fine structure of the retina and choroid in rat was fairly well preserved, and it was concluded that this method was more suitable for the observation of membraneous systems of the cell than that with conventional method. The purpose of this study is conducted to compare the fine structure of the outer retinal layers of turtle and frog by this technique with that of conventional method.
Materialsand Methods Turtles (Clemmys japonica) and frogs (Rana catesbeiana) were used. After enucleation, the retina was dissected from the choroid, cut into small pieces, and fixed in a 2.5% glutaraldehyde solution in 1/15 M phosphate buffer (pH 7.2) for 20--30 min in an icebox. Then, the specimens were washed with the same buffer for 3 - 5 h. They were immersed in a 1.0 M sucrose solution for 20 rain, cut into about 1.0 mm a sized blocks
2
N. Maekawa and K. Mizuno
and frozen directly with liquid nitrogen. After freezing, the specimen holder was attached quickly to the precooled LKB cryo-ultramicrotome. The sectioning was performed at -110~ specimen temperature and - 8 0 ~ knife temperature without any antifreeze through liquid nitrogen, respectively. The frozen sections were collected with droplet of a 2.3 M sucrose solution by the method of Tokuyasu (1973). Next, they were floated on a 0.5 or 1.0% phosphotungstic acid (PTA) solution at room temperature for staining. They were mounted on carbon-coated collodion grids, and were examined with a JEM-100 electron microscope. On the control observation of dense bodies in the pigment epithelium of turtle, the specimens were prefixed with a 2.5% glutaraldehyde in 1/15 M phosphate buffer (pH 7.2) for 20--30 min, and postfixed by 1% osminum tetroxide buffered with 1/15 M phosphate for 2 h. Then they were dehydrated with graded ethanol and embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate, and examined by the same method.
Results
The retinal pigment epithelium was filled with a large number of negatively stained, smooth-surfaced endoplasmic reticulum (SER) in all animals examined. In the turtle eye, mitochondria in the pigment epithelium were extraordinarily long and slender in shape, and their cristae were found arranged parallel to their long axis. They tended to run parallel to the plane of the Bruch's membrane. The membrane structures, including the cristae of mitochondria, were clearly negatively stained. However, numerous free ribosomes, which were confirmed in the control specimen by the conventional method, were obscure in the pigment epithelium. Myeloid bodies of turtle were observed in double flat-conical form and a biconvex lens shape. Myeloid bodies were situated mainly at the nuclear zone of the pigment epithelium, and less in number in the microvilli and near the cell border of the pigment epithelial cells. They represented clear lamellar configuration consisting of unit membranes of about 70 )~ in width. Individual discs of myeloid bodies were sometimes continuous at their margin with surrounding endoplasmic reticulum. Some myeloid bodies were in contact with the nuclear membrane of the pigment epithelium; and others were surrounded by large mitochondria in part. The interecellular space of the pigment epithelium was slightly opened, and measured 2 0 0 800 • in width; but the zonule occludens was tightly closed and the zonule adherens was positively stained with PTA (Fig. 1). Pigment granules, which were surrounded with positively stained halos, represented moderate positive staining, and were situated in the microvilli of the pigment epithelium. Round-shaped, numerous bodies, 0.5-1.0 # in diameter, were scattered from the nuclear to apical zone. Dense bodieswere, in general, enclosed with a single membrane, and demarcated from surrounding elements with positively stained halos around them. They included negatively stained, myelinlike, homogeneous (Fig. 2 A), and variously sized granular matrices (Figs. 2 B - D and 3 A). A peculiar, large, dense body was crescent in shape, and engulfed further smaller dense bodies in its matrix. Dense bodies engulfed in it had lost their single membrane, and their fine granular matrices were apparently continuous with the matrix of the mother dense body. On the other hand, membrane
Cryoultramicroscopy of Lower Vertebrate Retina
3
Fig. 1. Retinal pigment epithelium of turtle. Pigment granules (Pg), dense body (D), myeloid body (My), and mitochondria (M) are shown. Intercellular space of pigment epithelium is slightly opened, and zonula adherens shows positive image and zonnla occludens is tightly closed. X 14080
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N. Maekawa and K. Mizuno
Fig. 2 A--D. Dense bodies of turtle with different-shaped retinal pigment epithelium. They are demarcated from surroundings with positively stained halo, including myelinlike, homogeneous (A), and granular matrix (B, C, D). Limiting membrane disappears in part (B, D). A X 36 560, B X 27040, C X 24800, D X 37280
and granular structure of dense bodies observed by the conventional method was less distinct in comparison with those observed by cryoultramicroscopy (Fig. 3 B). Largely speaking, at least four types of dense bodies were identified from the structures in their matrix. A large granular matrix was sometimes continuous with surrounding SER tubules through a broken single membrane (Fig. 3 A). In some cases, a dense matrix was filled with a row of large granules outside and fine granules inside (Fig. 2 C and D). Some of the dense bodies were composed of myelin structures, and the membrane of the myelinlike matrix was about 80 A in width, being thicker than that of myeloid bodies, but thinner than that of the cone outer segment (Fig. 2 A). Homogenous matrices were, in general, included together with myelinlike matrices in the same dense body (Fig. 2 A) and also concentric configurations of myelinlike matrices were observed independently (Fig. 3 A). Single membranes of some dense bodies opened to connect either with SER or myeloid bodies (Fig. 2 A - D ) . Golgi apparatus were observed clearly with negatively stained vesicles and cisterna (Fig. 4). In the turtle eye, the ellipsoid of the inner segment was full of the atypical mitochondria situated large at center part and small at peripheral part. Their cristae did not show clubbed structures, which usually are observed by the conventional method, but dense granular structures instead. Several crystalline structures and a latticelike one were found in the mitochondria (Fig. 6). They were considered to be a lipidlike sub-
Cryoultrarnicroscopy of Lower Vertebrate Retina
5
Fig. 3. A Large dense body of retinal pigment epithelium of turtle. It includes several small dense bodies whose limiting membrane is entirely lost and concentric configurations of myelinlike matrix is shown. X 24000. B Dense bodies of retinal pigment epithelium of turtle by conventional method. X 32000
stance, but any transitional form from the lipidlike substance to oil droplet was not found in the present experiment. Oil droplet in the ellipsoid was not observed, but the corresponding part was vacant. Oil droplet and mitochondria were smoothly connected to each other. In the paraboloid of the inner segment, the glycogen-containing body was observed in the cortex, and aggregated glycogen particles were seen in tubules of the medulla which were stained negatively (Fig. 5). Numerous synaptic vesicles stained negatively were full in synaptic expansion of cone foot (Fig. 7). Synaptic ribbons were clearly observed as lamellar structures which were composed of two unit membrances, measuring about 140 • in each width, and a single membrane which was scarcely recognized. The positively stained area was observed between the synaptic ribbon and adjoining synaptic vesicles (Fig. 7 inset).
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Fig. 4. Golgi apparatus in retinal pigment epithelium of turtle. Golgi vesicles and cisternae are clearly negatively stained. X 32 000
Fig. 5. Paraboloid of inner segment of turtle. Glycogen-containing b o d y is s h o w n in cortex, and aggregated glycogen particles are scattered in negatively stained tubules of medulla. X 16 000
Cryoultramicroscopy o f Lower Vertebrate Retina
Fig. 6 A and B. Cone outer segment and ellipsoid o f inner segment o f turtle. Several crystalline (A) and latticelike structures (B) are found within atypical mitochondria. A X 1 7 3 6 0 , B X 29 360
Fig. 7. A. Cone food synaptic expansion of turtle. N u m e r o u s synaptic vesicles are stained negatively. X 6 800. B Synaptic ribbon in outer plexiform layer of frog. Synaptic ribbon consists of a two-unit m e m b r a n e and single m e m b r a n e . X 29 200
7
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Discussion
Technical problems which have been considered unavoidable in the field of cryoultramicroscopy were recently improved by Tokuyasu (1973), and fine structures are now fairly well preserved by his technique. By means of application of this new technique, our previous study (Maekawa and Mizuno, 1976) proved that this method is also useful for the study of structure of the rat retina, especially on its membrane structure. This fact was also confirmed in the present experiment on the fine structure of the retina of frog and turtle. Not only the cell membrane of the pigment epithelial cell, but also membranous continuity between myeloid body and tubules of SER and that between Golgi cisternae and vesicles, were well exemplified. The most interesting features of the pigment epithelial cell of turtle is the presence of numerous, altered, shaped, dense bodies which are identical with phagosomes. The smallest and most numerous of these particles have a very regular morphology and are strictly similar in appearance to the lysosomes seen by conventional electronmicroscopy. A part of the dense bodies are believed to be phagosomes, and they are usually spherical, about 0.5-1.0/~ in diameter, and consist of granular, homogenous, and myelinlike matrices, surrounded by single membranes with positively stained halos around them. These phagosomes are believed to be lysosome on their morphologic stand points. However, it is of particular interest to note that the prototype of lamellar inclusion bodies, such as fragments of the rod outer segment and fingerprint structures frequently observed in the pigment epithelium of higher mammalians, was not found in the pigment epithelial cell of turtles, but concentric configurations of myelinlike matrices in phagosomes were seen in the nuclear zone of the turtle pigment epithelium. This fact would suggest that renewal of the cone outer segment could occur in the cone retina. Though there has not been direct proof of engulfment of the cone outer segment into the pigment epithelium of turtle (Yamada, 1961), because a nocturnal turtle has been known to contain the rod outer segment in part (Walls, 1942), the origin of myelinlike matrices in some phagosomes may.be the rod outer segments. Myeloid bodies are fundamentally different from the Iamellar inclusion bodies because the former bodies are apparently derived from and continuous with SER and are not surrounded by a membrane (Porter and Yamada, 1968). Therefore, it would be wise to reach a final conclusion of that origin in some future study of the subject. Although phagosomes in the pigment epithelium are extremely varied in size, shape, and content, they all seem to be related because of the structural characteristics they share. The many transitional stages suggest that the different forms of the phagosomes may present a sequence in which large granular substances may degrade into fine or homogenous materials. Some large granular matrices are apparently continuous with SER and the single membranes are discontinuous at such parts - probably suggesting one of the end stages of lysosome particles. In addition, homogenous and fine granular matrices in the phagosomes may also indicate the end stage of lysosomal digestion, because they are sometimes continuous with the surroundings through a broken single membrane. Phagosomes, whose single membrane is wholly continuous and contains large granules in the outside of it and fine granules in the most inner part of it, may indicate a process of digestion with lysosomal enzymes. Features of a large crescent phagosome, which contains, and digests several small phagosomes, may apparently
Cryoultramicroscopy of Lower Vertebrate Retina
9
support the above assumption, because small phagosomes engulfed have definitely lost their single membrane, and demonstrate the degradation process of a large granular matrix into small and fine ones. However, because of the absence of other evidence (i.e., histochemistry of lysosomal enzymes), a final decision on whether or not this represents an actual evolving sequence in the pigment epithelium of turtles cannot be given as yet. Atypical mitochondria and oil droplets found in the ellipsoid in the lower vertebrate were classified into two types by Isikawa and Yamada (1969). Type 1 is similar to liquid droplets in the pigment epithelium, being stained homogenously in various densities. And type 2 is mitochondria at the scleral side of the ellipsoid, assumed as a precursor of oil droplets by Berger (1966). They emphasized that oil droplets of type 1 and 2 are entirely different in structure. A crystalline substance observed in the ellipsoid mitochondria is possibly identical with that reported by Ishikawa and Yamada (1966) as a dense amorphous material, which was regarded as a lipidlike substance by them. Again, this crystalline substance is also similar to the crystalloid substance found by Kuwabara (1975) in mitochondria of the ellipsoid of frogs during hibernation. Therefore, lipid crystalline may be fairly well preserved in its original form by only cryoultramicroscopy. Thus, it can be emphasized that one of the advantages of cryoultramicroscopy is better preservation of a greater number of synaptic vesicles in the synaptic end food of the cone rather than in that observed by the conventional method. Hogan et al. (1971) demonstrated synaptic ribbons composed of three dense layers approximately 120 A in each thickness, separated from each other by two electronless-dense layers of 40 A in thickness by the conventional method. Cryoultramicroscopic findings of synaptic ribbon of the cone end f o o t of turtles revealed, however, two negatively stained layers approximately 140 A in thickness and a single thin layer between them which was barely detectable. Whether the difference between Hogan's results and ours is due to a difference in species used or technique employed is a matter which cannot be decided at this time.
References Berger, E.R. : On the mitochondrial origin of oil drops in the retinal double cone inner segments. J. Ultrastruct. Res. 14, 143-157 (1966) Fernandes-Moran, H., Orville, A.D.: Electron microscopy of the ultrathin frozen sections of pollen grains. Science 116, 465-467 (1952) Hogan, M.J.,Alvarado, J.A., Weddell, J.E.: Histology of the human eye, pp. 451. PhiladelphiaLondon--Toronto: Saunders 1971 Ishikawa, T., Yamada, E.: Atypical mitochondria in the ellipsoid of the photoreceptor cells of vertebrate retinas, Invest. Ophthalrn. 8, 302- 316 (1969) Koda, N.: The preparation of ultrathin frozen sections of the retina in rat. Part I. Acta Soc. Ophthalm. Jap. 77, 1973-1981 (1973) Koda, N.: The preparation of ultrathin frozen sections of the retina in rat. Part II. Acta Soc. Ophthalm. Jap. 78, 324-331 (1974a) Koda, N.: The preparation of ultrathin frozen sections of the retina in rat. Part III. Acta Soc. Ophthalm. Jap. 78, 686-695 (1974b) Koda, N., Mizuno, K.: Preparation of ultratbin frozen sections of the retina. Part I. The photoreceptor stained with phosphotungstic acid. Jap. J. Ophthal. 18, 226-232 (1974) Kuwabara, T.: Cytologic changes of the retina and pigment epithelium during hibernation. Invest. Ophthalm. 14, 457-469 (1975)
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Maekawa, N., Mizuno, K.: Cryoultramicroscopy of the retina and choroid in rat. Albrecht v. Graefes Arch. klin. exp. Ophthalm. 200, 113-121 (1976) Porter, K.R., Yamada, E.: Studies on the endoplasmic reticulum. V its form and differentiation in pigment epithelial cells of the frog retina. J. Biophys. & Biochem. Cyto. 8 , 1 8 1 - 2 0 5 (1960) Tokuyasu, K.T.: A technique for ultracryotomy of all suspensions and tissues. J. Cell Biol. 57, 551-565 (1973) Walls, G.L.: The vertebrate eye and its adaptive radiation. Cranbrook Inst. Science (1942) Yamada, E.: The fine structure of the pigment epithelium in the turtle eye. In: Structure of the eye (G.K. Smelser, ed.), pp. 73-84. London: Academic Press 1961
Received July 22, 1976
Graefes Archiv for klinischeundexperimentelle
Ophthalmologie ,~ by Springer-Verlag 1976
Cryoultramicroscopy of Lower Vertebrate Retina Nobuo Maekawa and Katsuyoshi Mizuno Department of Ophthalmology, Tohoku University School of Medicine, Seiryo-cho, Sendai, 980 Japan Summary. Cryoultramicroscopy was carried out in the retina of lower vertebrates. Myeloid bodies of the pigment epithelium of turtles and synaptic vesicles and ribbons of frogs were stained negatively. Numerous altered, shaped, dense bodies, containing granular and myelinlike matrices, were found in the retinal pigment epithelium of turtle, and were thought to be lysosome. The advantages and disadvantages of this method are discussed.
Introduction Since the first report of cryoultramicroscopy by Fernandez-Moran and Orville (1952) had been presented, several authors (Koda, 1973, 1974 a, b; Koda and Mizuno, 1974) attempted to observe the retina by cryoultramicroscopy, but without satisfactory results. Thus, study of the fine structure of the retina with this technique was a difficult matter, because of its technical problems to preserve the ultrathin frozen section. The difficulties were partly overcome with the new technique, which improves preservation of the ultrathin frozen sections and improves the pick-up of sections by using sucrose (Tokuyasu, 1973). This new technique seems worthwhile from the principle and practice of cryoultramicroscopy. In our previous report (Maekawa and Mizuno, 1976), the fine structure of the retina and choroid in rat was fairly well preserved, and it was concluded that this method was more suitable for the observation of membraneous systems of the cell than that with conventional method. The purpose of this study is conducted to compare the fine structure of the outer retinal layers of turtle and frog by this technique with that of conventional method.
Materialsand Methods Turtles (Clemmys japonica) and frogs (Rana catesbeiana) were used. After enucleation, the retina was dissected from the choroid, cut into small pieces, and fixed in a 2.5% glutaraldehyde solution in 1/15 M phosphate buffer (pH 7.2) for 20--30 min in an icebox. Then, the specimens were washed with the same buffer for 3 - 5 h. They were immersed in a 1.0 M sucrose solution for 20 rain, cut into about 1.0 mm a sized blocks
2
N. Maekawa and K. Mizuno
and frozen directly with liquid nitrogen. After freezing, the specimen holder was attached quickly to the precooled LKB cryo-ultramicrotome. The sectioning was performed at -110~ specimen temperature and - 8 0 ~ knife temperature without any antifreeze through liquid nitrogen, respectively. The frozen sections were collected with droplet of a 2.3 M sucrose solution by the method of Tokuyasu (1973). Next, they were floated on a 0.5 or 1.0% phosphotungstic acid (PTA) solution at room temperature for staining. They were mounted on carbon-coated collodion grids, and were examined with a JEM-100 electron microscope. On the control observation of dense bodies in the pigment epithelium of turtle, the specimens were prefixed with a 2.5% glutaraldehyde in 1/15 M phosphate buffer (pH 7.2) for 20--30 min, and postfixed by 1% osminum tetroxide buffered with 1/15 M phosphate for 2 h. Then they were dehydrated with graded ethanol and embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate, and examined by the same method.
Results
The retinal pigment epithelium was filled with a large number of negatively stained, smooth-surfaced endoplasmic reticulum (SER) in all animals examined. In the turtle eye, mitochondria in the pigment epithelium were extraordinarily long and slender in shape, and their cristae were found arranged parallel to their long axis. They tended to run parallel to the plane of the Bruch's membrane. The membrane structures, including the cristae of mitochondria, were clearly negatively stained. However, numerous free ribosomes, which were confirmed in the control specimen by the conventional method, were obscure in the pigment epithelium. Myeloid bodies of turtle were observed in double flat-conical form and a biconvex lens shape. Myeloid bodies were situated mainly at the nuclear zone of the pigment epithelium, and less in number in the microvilli and near the cell border of the pigment epithelial cells. They represented clear lamellar configuration consisting of unit membranes of about 70 )~ in width. Individual discs of myeloid bodies were sometimes continuous at their margin with surrounding endoplasmic reticulum. Some myeloid bodies were in contact with the nuclear membrane of the pigment epithelium; and others were surrounded by large mitochondria in part. The interecellular space of the pigment epithelium was slightly opened, and measured 2 0 0 800 • in width; but the zonule occludens was tightly closed and the zonule adherens was positively stained with PTA (Fig. 1). Pigment granules, which were surrounded with positively stained halos, represented moderate positive staining, and were situated in the microvilli of the pigment epithelium. Round-shaped, numerous bodies, 0.5-1.0 # in diameter, were scattered from the nuclear to apical zone. Dense bodieswere, in general, enclosed with a single membrane, and demarcated from surrounding elements with positively stained halos around them. They included negatively stained, myelinlike, homogeneous (Fig. 2 A), and variously sized granular matrices (Figs. 2 B - D and 3 A). A peculiar, large, dense body was crescent in shape, and engulfed further smaller dense bodies in its matrix. Dense bodies engulfed in it had lost their single membrane, and their fine granular matrices were apparently continuous with the matrix of the mother dense body. On the other hand, membrane
Cryoultramicroscopy of Lower Vertebrate Retina
3
Fig. 1. Retinal pigment epithelium of turtle. Pigment granules (Pg), dense body (D), myeloid body (My), and mitochondria (M) are shown. Intercellular space of pigment epithelium is slightly opened, and zonula adherens shows positive image and zonnla occludens is tightly closed. X 14080
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N. Maekawa and K. Mizuno
Fig. 2 A--D. Dense bodies of turtle with different-shaped retinal pigment epithelium. They are demarcated from surroundings with positively stained halo, including myelinlike, homogeneous (A), and granular matrix (B, C, D). Limiting membrane disappears in part (B, D). A X 36 560, B X 27040, C X 24800, D X 37280
and granular structure of dense bodies observed by the conventional method was less distinct in comparison with those observed by cryoultramicroscopy (Fig. 3 B). Largely speaking, at least four types of dense bodies were identified from the structures in their matrix. A large granular matrix was sometimes continuous with surrounding SER tubules through a broken single membrane (Fig. 3 A). In some cases, a dense matrix was filled with a row of large granules outside and fine granules inside (Fig. 2 C and D). Some of the dense bodies were composed of myelin structures, and the membrane of the myelinlike matrix was about 80 A in width, being thicker than that of myeloid bodies, but thinner than that of the cone outer segment (Fig. 2 A). Homogenous matrices were, in general, included together with myelinlike matrices in the same dense body (Fig. 2 A) and also concentric configurations of myelinlike matrices were observed independently (Fig. 3 A). Single membranes of some dense bodies opened to connect either with SER or myeloid bodies (Fig. 2 A - D ) . Golgi apparatus were observed clearly with negatively stained vesicles and cisterna (Fig. 4). In the turtle eye, the ellipsoid of the inner segment was full of the atypical mitochondria situated large at center part and small at peripheral part. Their cristae did not show clubbed structures, which usually are observed by the conventional method, but dense granular structures instead. Several crystalline structures and a latticelike one were found in the mitochondria (Fig. 6). They were considered to be a lipidlike sub-
Cryoultrarnicroscopy of Lower Vertebrate Retina
5
Fig. 3. A Large dense body of retinal pigment epithelium of turtle. It includes several small dense bodies whose limiting membrane is entirely lost and concentric configurations of myelinlike matrix is shown. X 24000. B Dense bodies of retinal pigment epithelium of turtle by conventional method. X 32000
stance, but any transitional form from the lipidlike substance to oil droplet was not found in the present experiment. Oil droplet in the ellipsoid was not observed, but the corresponding part was vacant. Oil droplet and mitochondria were smoothly connected to each other. In the paraboloid of the inner segment, the glycogen-containing body was observed in the cortex, and aggregated glycogen particles were seen in tubules of the medulla which were stained negatively (Fig. 5). Numerous synaptic vesicles stained negatively were full in synaptic expansion of cone foot (Fig. 7). Synaptic ribbons were clearly observed as lamellar structures which were composed of two unit membrances, measuring about 140 • in each width, and a single membrane which was scarcely recognized. The positively stained area was observed between the synaptic ribbon and adjoining synaptic vesicles (Fig. 7 inset).
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N. Maekawa and K. Mizuno
Fig. 4. Golgi apparatus in retinal pigment epithelium of turtle. Golgi vesicles and cisternae are clearly negatively stained. X 32 000
Fig. 5. Paraboloid of inner segment of turtle. Glycogen-containing b o d y is s h o w n in cortex, and aggregated glycogen particles are scattered in negatively stained tubules of medulla. X 16 000
Cryoultramicroscopy o f Lower Vertebrate Retina
Fig. 6 A and B. Cone outer segment and ellipsoid o f inner segment o f turtle. Several crystalline (A) and latticelike structures (B) are found within atypical mitochondria. A X 1 7 3 6 0 , B X 29 360
Fig. 7. A. Cone food synaptic expansion of turtle. N u m e r o u s synaptic vesicles are stained negatively. X 6 800. B Synaptic ribbon in outer plexiform layer of frog. Synaptic ribbon consists of a two-unit m e m b r a n e and single m e m b r a n e . X 29 200
7
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N. Maekawa and K. Mizuno
Discussion
Technical problems which have been considered unavoidable in the field of cryoultramicroscopy were recently improved by Tokuyasu (1973), and fine structures are now fairly well preserved by his technique. By means of application of this new technique, our previous study (Maekawa and Mizuno, 1976) proved that this method is also useful for the study of structure of the rat retina, especially on its membrane structure. This fact was also confirmed in the present experiment on the fine structure of the retina of frog and turtle. Not only the cell membrane of the pigment epithelial cell, but also membranous continuity between myeloid body and tubules of SER and that between Golgi cisternae and vesicles, were well exemplified. The most interesting features of the pigment epithelial cell of turtle is the presence of numerous, altered, shaped, dense bodies which are identical with phagosomes. The smallest and most numerous of these particles have a very regular morphology and are strictly similar in appearance to the lysosomes seen by conventional electronmicroscopy. A part of the dense bodies are believed to be phagosomes, and they are usually spherical, about 0.5-1.0/~ in diameter, and consist of granular, homogenous, and myelinlike matrices, surrounded by single membranes with positively stained halos around them. These phagosomes are believed to be lysosome on their morphologic stand points. However, it is of particular interest to note that the prototype of lamellar inclusion bodies, such as fragments of the rod outer segment and fingerprint structures frequently observed in the pigment epithelium of higher mammalians, was not found in the pigment epithelial cell of turtles, but concentric configurations of myelinlike matrices in phagosomes were seen in the nuclear zone of the turtle pigment epithelium. This fact would suggest that renewal of the cone outer segment could occur in the cone retina. Though there has not been direct proof of engulfment of the cone outer segment into the pigment epithelium of turtle (Yamada, 1961), because a nocturnal turtle has been known to contain the rod outer segment in part (Walls, 1942), the origin of myelinlike matrices in some phagosomes may.be the rod outer segments. Myeloid bodies are fundamentally different from the Iamellar inclusion bodies because the former bodies are apparently derived from and continuous with SER and are not surrounded by a membrane (Porter and Yamada, 1968). Therefore, it would be wise to reach a final conclusion of that origin in some future study of the subject. Although phagosomes in the pigment epithelium are extremely varied in size, shape, and content, they all seem to be related because of the structural characteristics they share. The many transitional stages suggest that the different forms of the phagosomes may present a sequence in which large granular substances may degrade into fine or homogenous materials. Some large granular matrices are apparently continuous with SER and the single membranes are discontinuous at such parts - probably suggesting one of the end stages of lysosome particles. In addition, homogenous and fine granular matrices in the phagosomes may also indicate the end stage of lysosomal digestion, because they are sometimes continuous with the surroundings through a broken single membrane. Phagosomes, whose single membrane is wholly continuous and contains large granules in the outside of it and fine granules in the most inner part of it, may indicate a process of digestion with lysosomal enzymes. Features of a large crescent phagosome, which contains, and digests several small phagosomes, may apparently
Cryoultramicroscopy of Lower Vertebrate Retina
9
support the above assumption, because small phagosomes engulfed have definitely lost their single membrane, and demonstrate the degradation process of a large granular matrix into small and fine ones. However, because of the absence of other evidence (i.e., histochemistry of lysosomal enzymes), a final decision on whether or not this represents an actual evolving sequence in the pigment epithelium of turtles cannot be given as yet. Atypical mitochondria and oil droplets found in the ellipsoid in the lower vertebrate were classified into two types by Isikawa and Yamada (1969). Type 1 is similar to liquid droplets in the pigment epithelium, being stained homogenously in various densities. And type 2 is mitochondria at the scleral side of the ellipsoid, assumed as a precursor of oil droplets by Berger (1966). They emphasized that oil droplets of type 1 and 2 are entirely different in structure. A crystalline substance observed in the ellipsoid mitochondria is possibly identical with that reported by Ishikawa and Yamada (1966) as a dense amorphous material, which was regarded as a lipidlike substance by them. Again, this crystalline substance is also similar to the crystalloid substance found by Kuwabara (1975) in mitochondria of the ellipsoid of frogs during hibernation. Therefore, lipid crystalline may be fairly well preserved in its original form by only cryoultramicroscopy. Thus, it can be emphasized that one of the advantages of cryoultramicroscopy is better preservation of a greater number of synaptic vesicles in the synaptic end food of the cone rather than in that observed by the conventional method. Hogan et al. (1971) demonstrated synaptic ribbons composed of three dense layers approximately 120 A in each thickness, separated from each other by two electronless-dense layers of 40 A in thickness by the conventional method. Cryoultramicroscopic findings of synaptic ribbon of the cone end f o o t of turtles revealed, however, two negatively stained layers approximately 140 A in thickness and a single thin layer between them which was barely detectable. Whether the difference between Hogan's results and ours is due to a difference in species used or technique employed is a matter which cannot be decided at this time.
References Berger, E.R. : On the mitochondrial origin of oil drops in the retinal double cone inner segments. J. Ultrastruct. Res. 14, 143-157 (1966) Fernandes-Moran, H., Orville, A.D.: Electron microscopy of the ultrathin frozen sections of pollen grains. Science 116, 465-467 (1952) Hogan, M.J.,Alvarado, J.A., Weddell, J.E.: Histology of the human eye, pp. 451. PhiladelphiaLondon--Toronto: Saunders 1971 Ishikawa, T., Yamada, E.: Atypical mitochondria in the ellipsoid of the photoreceptor cells of vertebrate retinas, Invest. Ophthalrn. 8, 302- 316 (1969) Koda, N.: The preparation of ultrathin frozen sections of the retina in rat. Part I. Acta Soc. Ophthalm. Jap. 77, 1973-1981 (1973) Koda, N.: The preparation of ultrathin frozen sections of the retina in rat. Part II. Acta Soc. Ophthalm. Jap. 78, 324-331 (1974a) Koda, N.: The preparation of ultrathin frozen sections of the retina in rat. Part III. Acta Soc. Ophthalm. Jap. 78, 686-695 (1974b) Koda, N., Mizuno, K.: Preparation of ultratbin frozen sections of the retina. Part I. The photoreceptor stained with phosphotungstic acid. Jap. J. Ophthal. 18, 226-232 (1974) Kuwabara, T.: Cytologic changes of the retina and pigment epithelium during hibernation. Invest. Ophthalm. 14, 457-469 (1975)
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N. Maekawa and K. Mizuno
Maekawa, N., Mizuno, K.: Cryoultramicroscopy of the retina and choroid in rat. Albrecht v. Graefes Arch. klin. exp. Ophthalm. 200, 113-121 (1976) Porter, K.R., Yamada, E.: Studies on the endoplasmic reticulum. V its form and differentiation in pigment epithelial cells of the frog retina. J. Biophys. & Biochem. Cyto. 8 , 1 8 1 - 2 0 5 (1960) Tokuyasu, K.T.: A technique for ultracryotomy of all suspensions and tissues. J. Cell Biol. 57, 551-565 (1973) Walls, G.L.: The vertebrate eye and its adaptive radiation. Cranbrook Inst. Science (1942) Yamada, E.: The fine structure of the pigment epithelium in the turtle eye. In: Structure of the eye (G.K. Smelser, ed.), pp. 73-84. London: Academic Press 1961
Received July 22, 1976
