Anat Embryol (1992) 186: 371-377
Anatomyand
Embryology
9 Springer-Verlag1992
Retinal pigment epithelial fine structure in the velvet cichlid (Astronotus ocellatus) Charlie R. Braekevelt Department of Anatomy, The University of Manitoba, 730 William Avenue, Winnipeg, Manitoba R3E 0W3, Canada Accepted June 7, 1992
Summary. The morphology of the retinal pigment epithelium (RPE), choriocapillaris and Bruch's membrane (complexus basalis) have been studied by light and electron microscopy in the velvet cichlid (Astronotus ocellatus). The RPE is composed of a single layer of large columnar cells. The basal (scleral) border of these cells is minimally infolded, whereas the apical (vitreal) surface displays numerous pigment-laden processes which in light-adaptation surround both rod and cone outer segments. Laterally the RPE cells are joined by a series of basally located tight junctions. Wandering phagocytes are a constant feature within this epithelial membrane. The R P E cells display a large, vesicular nucleus, numerous mitochondria, much smooth endoplasmic reticulum, polysomes, myeloid bodies, phagosomes and melanosomes. Rough endoplasmic reticulum is relatively scarce within these cells. Although only light-adapted specimens were examined, it is thought that the melanosomes are capable of extensive retinomotor movement. The endothelium of the choriocapillaris facing Bruch's membrane is typically very thin but shows few fenestrations. Bruch's membrane is typical of other teleost species in that it is composed of only three layers.
of the elongate photoreceptor outer segments (Bernstein 1961; Enoch 1979) 3. The internal adhesion of the neurosensory retina (Zinn and Benjamin-Henkin 1979) 4. The storage and modification of vitamin A precursors of the visual pigments (Young and Bok 1970) 5. The selective transport of materials to and from the photoreceptor cells (Kroll and Machemer 1968; Steinberg and Miller 1973) As a consequence of the RPE being involved in such a number of important functions, the R P E region has been investigated in a variety of animals with a multiplicity of techniques. Morphological studies have shown that while this region is quite similar in all vertebrates investigated to date, generic differences are always present (Nguyen-Legros 1978; Kuwabara 1979; Braekevelt 1980a, 1982, 1984, 1986, 1988, 1990). In this report the fine structure of the retinal epithelium (RPE), choriocapillaries and Bruch's membrane or complexus basalis is described in the velvet cichlid (Astronotus ocellatus) and compared and contrasted with that observed in other vertebrate species.
Key words: Retinal pigment epithelium (RPE) - Electron microscopy Teleost - Astronatus ocellatus
Materials and methods
Introduction The retinal pigment epithelium (RPE) is the outermost layer of the neural retina, and along with the choriocapillaris and Bruch's membrane (complexus basalis) is involved in several processes crucial to the proper functioning o f the photoreceptors, and hence to vision itself. Amongst the best understood functions of the RPE are: 1. The phagocytosis and subsequent lysosomal degradation of shed photoreceptor outer segment discs (Bok and Young 1979) 2. The architectural support and effective orientation
For this study the eyes from four healthy, light-adapted adult velvet cichlids (also known as Oscar's cichlid) (Astronotus ocellatus) were examined by light and electron microscopy. Velvetcichlids are pugnacious, territorial fish which are diurnal and very visually oriented. The fish were obtained from local suppliers and held in aerated aquaria for several days prior to sampling. The fish were decapitated and the eyeballs quickly removed, opened at the equator and fixed for 5 h in 5% gluteraldehyde buffered to pH 7.3 with 0.1 M Sorensen's phosphate buffer at 4~ C. The posterior half of the eyeball was then removed, washed in 5% sucrose in 0.1 M Sorensen's buffer (pH 7.3) and cut into pieces less than 1 mm 2. The tissue was then postfixed for 2 h in 1% osmium tetroxide in the same phosphate buffer, dehydrated up through graded ethanols to methanol and then propylene oxide, and embedded in Araldite. Pieces of plastic-embedded tissue were subsequently reoriented to desired angles (by means of a wax mount) and thick sections
372 (0.5 gm) were cut, stained with toluidine blue and examined by light microscopy.Thin sections (50-60 nm) of selectedareas were then cut, collected on copper grids and stained in aqueous uranyl acetate and lead citrate, and examined and photographed in a Philips EM201 transmissionelectronmicroscope. Results
The retinal epithelium (RPE) of the velvet cichlid (Astronotus ocellatus) consists of a single layer of columnar cells measuring about 25 ~tm in height (Figs. 1, 3). Basically (sclerally) these cells are minimally infolded (Figs. 1, 3, 7, 8) while apically (vitreally) they display numerous finger-like processes (Figs. 2, 9). In the lightadapted condition, these apical processes are filled with melanosomes and surround and isolate the rod inner and outer segments as well as cone outer segments from one another (Figs. 1, 2, 9). These apical processes also reach to the cone inner segments, but they do not surround and isolate this portion of the cone photoreceptors (Fig. 2). Laterally these retinal epithelial cells display relatively smooth borders, and are joined basally by tight junctions to form a continuous epithelial sheet (Figs. 1, 3, 5, 8). Internally the RPE cells display a single large vesicular nucleus usually located in the midregion of the cell (Figs. 1, 4). Numerous round to oval or oblong mitochondria are predominantly located in the basal region of these epithelial cells (Figs. 3, 5, 6, 8). These mitochondria display a very electron-dense matrix in light-adaptation (Figs. 3, 5, 6). Smooth endoplasmic reticulum (SER) is widespread, while rough endoplasmic reticulum (RER) is very scarce (Figs. 2, 3, 4, 6). Polysomes are however widespread (Figs. 5, 6, 7, 8). Lysosome-like bodies and small (1.0 gm) lipid bodies were also present within these epithelial cells, with the lipid bodies being mainly located in the basal regions (Figs. 1, 3, 5, 6, 8). Melanosomes, which in this species are rod-shaped and measure up to 5 or 6 gm in length (Fig. 9), are in the light-adapted state predominantly located in the apical region of the cell bodies and within the apical processes (Figs. 1, 2, 4, 9). Within the apical processes they are aligned with the long axes of these processes and the photoreceptor cells (Figs. 2, 9). Myeloid bodies are a constant feature of these retinal epithelial cells, and in the light-adapted state are small and mostly located in the apical region of these cells (Figs. 1, 2). Phagosomes of shed outer segment material, while not abundant in these light-adapted specimens, are still present (Figs. 3, 5, 7). These phagosomes are often difficult to differentiate from the viable rod outer segments, which in light-adaptation also reach deep into the RPE cells (Figs. 1, 7, 8). As has been noted in most other teleost species, wandering phagocytes are a consistent feature of the retinal epithelial layer (Fig. 3). In these light-adapted specimens the wandering phagocytes were always located amongst the rod outer segments, and they usually contained phagosomes of outer segment material as well as melanosomes (Fig. 3). Bruch's membrane or complexus basalis in this species is typical of that noted in other teleosts in that it
is a trilaminate structure (Figs. 1, 5, 7, 8). It averages about 0.75 ~tm in thickness, and is composed of the basal lamina of the retinal epithelium and the: basal lamina of the choriocapillaris endothelium enclosing a thick layer of collagenous fibrils (Figs. 6, 7, 8). The choriocapillaris forms a single layer of large calibre capillaries immediately adjacent to the choroidal aspect of the complexus basalis (Figs. 1, 6, 7). The endothelium facing Bruch's membrane, while extremely thin, is only minimally fenestrated (Figs. 7, 8). Discussion
The retinal epithelium (RPE) of the velvet cichlid (Astronotus ocellatus) is basically similar to that described in other vertebrates, with modifications that seem to be specific to teleostean species (Rodieck 1973; NguyenLegros 1978; Kuwabara 1979; Braekevelt 1983, 1985a, 1989, 1991). As in all described species, the retinal epithelium in the velvet cichlid consists of a single layer of cells. In teleosts in general, the RPE cells are normally columnar, while in mammals reptiles and birds they tend to be cuboidal, and in marsupials and amphibians they are usually more squamous in shape (Braekevelt 1980a, 1982, 1984, 1988, 1990, 1991). The basal (scleral) aspect of the RPE of most vertebrate species is extensively infolded. This may indicate enhanced transport from the choriocapillaris (Dowling and Gibbons 1962; Bernstein and Hollenberg 1965; Bok 1985). Teleost species on the other hand normally show only minimal infolding in this region (Okuda 1962; Braekevelt 1980, 1982a). This is thought to be due to the presence of a choroid gland, which has been shown to maintain a high oxygen pressure, and presumably lessens the requirements of oxygen transport by the RPE in teleosts (Wittenberg and Wittenberg 1974). Apically (vitreally) retinal epithelial cells normally display numerous processes which serve to support the photoreceptor outer segment structurally (Bernstein 1961), as well as orienting them most effectively to the incoming light (Enoch 1979). These apical processes are also important in the internal adhesion required between the RPE and the photoreceptors (Zinn and BenjaminHenkind 1979). In addition, the intimate relationship between the apical processes of the RPE cells and the photoreceptors serves to facilitate the movement of materials between these cells (Dowling 1960; Steinberg and Wood 1974). The lateral borders of the retinal epithelial cells are normally relatively smooth, and are always joined by a series of tight junctions (Kuwabara 1979). It has been shown that these tight junctions (collectively forming Verhoeff's membrane) provide an effective barrier to the intercellular movement of materials, and hence form part of the blood-ocular barrier (Zinn and BenjaminHenkind 1979). Internally, RPE cells invariably display a large vesicular nucleus and an abundance of cell organelles (NguyenLegros 1978; Kuwabara 1979; Braekevelt 1988, 1990).
Fig. 1. Low power electron micrograph of the retinal epithelium of the velvet cichlid. The choriocapillaris (CC), rod outer segments (ROS) and retinal epithelial nuclei (iV) are indicated • 6,400 In all micrographs the scale bar equals 1 gm
Fig. 2. Electron micrograph of the apical region of the RPE layer. Melanosomes (M)~ myeloid bodies (MY) as well as cone inner (CIS) and outer (COS) segments are all labelled, x 8,600
Fig. 3. Electron micrograph to iUustrate a wandering phagocyte (WP), numerous mitochondria (Mi) and a phagosome (Ph) of outcr segment material. Bruch's membrane (B) is also indicated. • ]2,800
Fig. 4. Electron micrograph to indicate a b u n d a n t smooth cndoplasmic reticulum (SER), a rctinal epithelial cell nucleus (N) and a phagosome (Ph). x 13,200 Fig. 5. Electron micrograph of the R P E layer to illustrate mitochondria (M0, a cell junction (J) and a phagosome (Ph). x ]2,6~00
Fig. 6. Electron micrograph to indicate lipid droplets (L), mitochondria (Mi) and a rod outer segment (ROS). Bruch's membrane (B) is also indicated. • 13,500
Fig. 8. Electron micrograph to illustrate a lipid droplet (L), melanosomes (M) and a cell junction (3). Bruch's membrane (B) is also indicated, x 13,000
Fig. 7. Electron micrograph to indicate the lack of basal infoldings and relatively few fenestrations in the choriocapillaris (CC) endothelium. A cell junction (J) and a rod outer segment (ROS) are also labelled, x 15,800
Fig. 9. Electron micrograph to illustrate the rod-like shape of melanosomes (M) within the apical processes of the RPE. A rod photoreceptor (R) is also labelied, x 14,400
376 As has been noted in other species, smooth endoplasmic reticulum (SER) is abundant within RPE cells, while rough endoplasmic reticulum (RER) is not. The relative scarcity of R E R in these cells would indicate that little protein is being produced for extracellular destinations, while the abundance of polysomes indicates that intracellular protein requirements are normal. An abundance of SER is c o m m o n to cells involved in lipid biosynthesis (Enders 1962), and it is certainly well established that the RPE is heavily involved in the storage, transport and esterification of vitamin A (Berman 1979; Zinn and Benjamin-Henkind 1979). The presence of lysosomes in this epithelial layer is also to be expected, as one o f the most important functions of this layer is the phagocytosis and lysosomal degradation of outer segment discs (Young and Bok 1969; Young 1978). The relative scarcity of phagosomes within the RPE cells of this fish in the light-adapted state is probably due to the sampling time, as it is known that rods shed soon after lights are on and cones shed after lights are off (Young 1978). Myeloid bodies are a fairly constant feature within the R P E cells o f lower vertebrates which show retinomotor movements of the melanosomes (Nicol et al. 1975; Braekevelt 1980a, 1982). They normally appear as stacked SER membranes (Kuwabara 1979) while in avian species they may have ribosomes on their outer surface (Braekevelt 1984, 1990). They have been implicated as sites for storage of lipids prior to esterification (Yorke and Dickson 1984, 1985) and as the organelle that triggers photomechanical movements (Porter and Yamada 1960; Braekevelt 1982) but the function of myeloid bodies remains uncertain. The melanosomes within the RPE cells of the velvet cichlid are in the light-adapted state more numerous in the apical region of the cell body and within the apical processes, where they surround and isolate both rod and cone photoreceptor outer segments. While only lightadapted specimens were examined in this study, judging by the abundance and location of the melanosomes, retinomotor responses are thought to occur in the velvet cichlid, as is reported for all teleost species (Walls 1942). The wandering phagocytes noted amongst the RPE cells in the velvet cichlid are thought to be a normal, non-pathological feature of the teleost retina (Braekevelt 1980b, 1985b). As they are often seen to contain phagosomes of outer segment material they are probably involved in the phagocytosis and degradation of outer segment discs, as are the attached RPE ceils. Teleosts in general have very long outer segments, and retinomotor responses of the photoreceptors are normally rapid and extensive. These factors may cause an accelerated rate and/or volume of shed outer segment discs, and hence the wandering phagocytes may be supplementing the RPE cells in the phagocytosis of this material. O ' D a y and Young (1978) have indicated that in the goldfish these wandering phagocytes are much more numerous during the period of peak disc-shedding, which would tend to support their presumed role of aiding the attached RPE cells to remove outer segment debris. Bruch's membrane or complexus basalis in mammalian species is invariably pentalaminate, and displays a
central elastic layer (lamina densa), which is absent in teleost species, to give a trilaminate structure (Braekevelt 1980a, 1986, 1988). In avian species, while the lamina densa is present, it is normally very poorly represented (Braekevelt 1984, 1988, 1990). The significance (if any) of these phylogenetic variations within Bruch's membrane is at present unknown. The choriocapillaris in all described species is composed of a single layer of large calibre capillaries (Walls 1942; Rodieck 1973; Kuwabara 1979). With the exception of teleosts, the endothelium facing the complexus basalis is highly fenestrated, indicating the movement of large quantities of material across this endothelium (Bernstein and Hollenberg 1965). In teleosts the presence of a choroid gland which is important in the maintenance of a high oxygen pressure is probably responsible for the reduction of the number of fenestrations of the choriocapillaris endothelium, as well as the reduced number of basal infoldings, two features that are characteristic of teleost species (Braekevelt 1980a, 1985a).
Acknowledgements. The excellent technical assistance of D.M. Love and R. Simpson is gratefully acknowledged. This work was supported in part by funds from the Natural Sciences and Engineering Research Council (NSERC) and the Medial Research Council (MRC) of Canada. References
Berman ER (1979) Biochemistry of the retinal pigment epithelium. In: Zinn KM, Marmor MF (eds) The retinal pigment epithelium. Harvard University Press, Cambridge, pp 83-102 Bernstein M H (1961) Functional architecture of the retinal epithelium. In: Smelser GK (ed) The structure of the eye. Academic Press, New York, pp 139-150 Bernstein MH, Hotlenberg MJ (1965) Movement of electronopaque tracers through the capillaries of the retina. In : Rohen JM (ed) The structure of the eye. II. Schattauer, Stuttgart, pp 129-138 Bok D (1985) Retinal photoreceptor-pigment epithelial interactions. Invest Ophthalmol Vis Sci 26:1659-1694 Bok D, Young RW (1979) Phagocytic properties of the retinal pigment epithelium. In: Zinn KM, Marmor MF (eds) The retinal pigment epithelium. Harvard University Press, Cambridge, Mass., pp 148-174 Braekeveit CR (1980a) Fine structure of the retinal repithelium and tapetum lucidum in the giant danio (Danio malabaricus) (Teleost). Anat Embryol 158 : 317-328 Braekevelt CR (1980b) Wandering phagocytes at the retinal epithelium - photoreceptor interface in the teieost retina. Vision Res 20:495~499 Braekevelt CR (1982) Fine structure of the retinal epithelium and retinal tapetum lucidum of the goldeye (Hiodon alosoides). Anat Embryol 164 :287-302 Braekevelt CR (1983) Fine structure of the choriocapillaris, Bruch's membrane and retinal epithelium in the sheep. Anat Embryol 166 :415-425 Braekevelt CR (1984) Retinal pigment epithelial fine structure in the nighthawk (Chordeiles minor). Ophthalmologica 188:222231 Braekevelt CR (1985a) Fine structure of the retinal pigment epithelia1 region of the archerfish (Toxotes jaculatrix). Ophthalmol Res 17:221 229 Braekevelt CR (1985b) Further observations on the presence of wandering phagocytes within the teleostean retina. Anat Anz 160 : 45-54
377 Braekevelt CR (1986) Fine structure of the choriocapillaris Bruch's membrane and retinal epithelium of the cow. Anat Histol Embryol 15:205-214 Braekevelt CR (1988) Retinal epithelial fine structure in the vervet monkey (Cercopithecus aethiops). Histol Histopathol 3 : 33-38 Braekevelt CR (1989) Retinal pigment epithelial fine structure in the bobtail goanna (Tiliqua rugosa). Histol Histopathol 4:295 300 Braekevelt CR (1990) Fine structure of the retinal pigment epithelium of the mallard duck (Anas platyrhynchos). Histol Histopathol 5:133-138 Braekevelt CR (1991) Retinal epithelial fine structure in the southern fiddler ray (Trygonorhinafasciata). Histol Histopathol (in press) DoMing JE (1960) Chemistry of visual adaptation in the rat. Nature 188:114-118 Dowling JE, Gibbons IR (1962) Fine structure of the pigment epithelium in the albino rat. J Ceil Biol 14:459-474 Enders AC (1962) Observations on the fine structure of lutein cells. J Cell Biol 12:101-113 Enoch JM (1979) Vertebrate receptor optics and orientation. Doc Ophthalmol 48 : 373 388 Kroll AJ, Machemer R (1968) Experimental retinal detachment in the owl monkey. III. Electron microscopy of retina and pigment epithelium. Am J Ophthalmol 66:410-427 Kuwabara T (1979) Species differences in the retinal pigment epithelium. In: Zinn KM, Marmor MF (eds) The retinal pigment epithelium. Harvard University Press, Cambridge, Mass., pp 58-82 Nicol JAC, Zyzmar ES, Thurston EL, Wang RT (1975) The tapeturn lucidum in the eyes of cusk-eels (Ophidiidae). Can J Zool 53:1063-1079 Nguyen-Legros J (1978) Fine structure of the pigment epithelium in the vertebrate retina. Int Rev Cytol [Suppl] 7:287-328 O'Day WT, Young RW (1978) Rhythmic daily shedding of outer segment membranes by visual cells in the goldfish. J Cell Biol 76 : 593-604
Okuda K (1962) Electron microscopic observations of the retinal pigment epithelium of vertebrate animals. Jpn J Ophthalmol 6:76 87 Porter KR, Yamada E (1960) Studies on the endoplasmic reticulure. V. Its form and differentiation in pigment epithelial cells of the frog retina. J Biophys Biochem Cytol 8:181-205 Rodieck RW (1973) The vertebrate retina: principles of structure and function. Freeman, San Francisco Steinberg RH, Miller S (1973) Aspects of electrolyte transport in frog pigment epithelium. Exp Eye Res 16:365 372 Steinberg RH, Wood I (1974) Pigment epithelial cell ensheathment of cone outer segments in the retina of the domestic cat. Proc R Soc Lond [Biol] 187:461-478 Walls GL (1942) The vertebrate eye and its adaptive radiation. Cranbrook Press, Bloomfield Hills Wittenberg JB, Wittenberg BA (1974) The choroid rete mirabile of the fish eye. I. Oxygen secretion and structure: comparison with the swim bladder rete mirabile. Biol Bull 146:116-136 Yorke MA, Dickson DH (1984) Diurnal variations in myeloid bodies of the newt retinal pigment epithelium. Cell Tissue Res 235:177 186 Yorke MA, Dickson DH (1985) A cytochemical study of myeloid bodies in the retinal pigment epithelium of the newt Notophthalmus viridescens. Cell Tissue Res 240 : 641-648 Young RW (1978) Visual cells, daily rhythms and vision research. Vision Res 18:573-578 Young RW, Bok D (1969) Participation of the retinal pigment epithelium in the rod outer segment renewal process. J Cell Biol 42:392-403 Young RW, Bok D (1970) Autoradiographic studies on the metabolism of the retinal pigment epithelium. Invest Ophthalmol 9:524-536 Zinn KM, Benjamin-Henkind JV (1979) Anatomy of the human retinal pigment epithelium. In: Zinn KM, Marmor MF (eds) The retinal pigment epithelium. Harvard University Press, Cambridge Mass., pp 3-31
Anatomyand
Embryology
9 Springer-Verlag1992
Retinal pigment epithelial fine structure in the velvet cichlid (Astronotus ocellatus) Charlie R. Braekevelt Department of Anatomy, The University of Manitoba, 730 William Avenue, Winnipeg, Manitoba R3E 0W3, Canada Accepted June 7, 1992
Summary. The morphology of the retinal pigment epithelium (RPE), choriocapillaris and Bruch's membrane (complexus basalis) have been studied by light and electron microscopy in the velvet cichlid (Astronotus ocellatus). The RPE is composed of a single layer of large columnar cells. The basal (scleral) border of these cells is minimally infolded, whereas the apical (vitreal) surface displays numerous pigment-laden processes which in light-adaptation surround both rod and cone outer segments. Laterally the RPE cells are joined by a series of basally located tight junctions. Wandering phagocytes are a constant feature within this epithelial membrane. The R P E cells display a large, vesicular nucleus, numerous mitochondria, much smooth endoplasmic reticulum, polysomes, myeloid bodies, phagosomes and melanosomes. Rough endoplasmic reticulum is relatively scarce within these cells. Although only light-adapted specimens were examined, it is thought that the melanosomes are capable of extensive retinomotor movement. The endothelium of the choriocapillaris facing Bruch's membrane is typically very thin but shows few fenestrations. Bruch's membrane is typical of other teleost species in that it is composed of only three layers.
of the elongate photoreceptor outer segments (Bernstein 1961; Enoch 1979) 3. The internal adhesion of the neurosensory retina (Zinn and Benjamin-Henkin 1979) 4. The storage and modification of vitamin A precursors of the visual pigments (Young and Bok 1970) 5. The selective transport of materials to and from the photoreceptor cells (Kroll and Machemer 1968; Steinberg and Miller 1973) As a consequence of the RPE being involved in such a number of important functions, the R P E region has been investigated in a variety of animals with a multiplicity of techniques. Morphological studies have shown that while this region is quite similar in all vertebrates investigated to date, generic differences are always present (Nguyen-Legros 1978; Kuwabara 1979; Braekevelt 1980a, 1982, 1984, 1986, 1988, 1990). In this report the fine structure of the retinal epithelium (RPE), choriocapillaries and Bruch's membrane or complexus basalis is described in the velvet cichlid (Astronotus ocellatus) and compared and contrasted with that observed in other vertebrate species.
Key words: Retinal pigment epithelium (RPE) - Electron microscopy Teleost - Astronatus ocellatus
Materials and methods
Introduction The retinal pigment epithelium (RPE) is the outermost layer of the neural retina, and along with the choriocapillaris and Bruch's membrane (complexus basalis) is involved in several processes crucial to the proper functioning o f the photoreceptors, and hence to vision itself. Amongst the best understood functions of the RPE are: 1. The phagocytosis and subsequent lysosomal degradation of shed photoreceptor outer segment discs (Bok and Young 1979) 2. The architectural support and effective orientation
For this study the eyes from four healthy, light-adapted adult velvet cichlids (also known as Oscar's cichlid) (Astronotus ocellatus) were examined by light and electron microscopy. Velvetcichlids are pugnacious, territorial fish which are diurnal and very visually oriented. The fish were obtained from local suppliers and held in aerated aquaria for several days prior to sampling. The fish were decapitated and the eyeballs quickly removed, opened at the equator and fixed for 5 h in 5% gluteraldehyde buffered to pH 7.3 with 0.1 M Sorensen's phosphate buffer at 4~ C. The posterior half of the eyeball was then removed, washed in 5% sucrose in 0.1 M Sorensen's buffer (pH 7.3) and cut into pieces less than 1 mm 2. The tissue was then postfixed for 2 h in 1% osmium tetroxide in the same phosphate buffer, dehydrated up through graded ethanols to methanol and then propylene oxide, and embedded in Araldite. Pieces of plastic-embedded tissue were subsequently reoriented to desired angles (by means of a wax mount) and thick sections
372 (0.5 gm) were cut, stained with toluidine blue and examined by light microscopy.Thin sections (50-60 nm) of selectedareas were then cut, collected on copper grids and stained in aqueous uranyl acetate and lead citrate, and examined and photographed in a Philips EM201 transmissionelectronmicroscope. Results
The retinal epithelium (RPE) of the velvet cichlid (Astronotus ocellatus) consists of a single layer of columnar cells measuring about 25 ~tm in height (Figs. 1, 3). Basically (sclerally) these cells are minimally infolded (Figs. 1, 3, 7, 8) while apically (vitreally) they display numerous finger-like processes (Figs. 2, 9). In the lightadapted condition, these apical processes are filled with melanosomes and surround and isolate the rod inner and outer segments as well as cone outer segments from one another (Figs. 1, 2, 9). These apical processes also reach to the cone inner segments, but they do not surround and isolate this portion of the cone photoreceptors (Fig. 2). Laterally these retinal epithelial cells display relatively smooth borders, and are joined basally by tight junctions to form a continuous epithelial sheet (Figs. 1, 3, 5, 8). Internally the RPE cells display a single large vesicular nucleus usually located in the midregion of the cell (Figs. 1, 4). Numerous round to oval or oblong mitochondria are predominantly located in the basal region of these epithelial cells (Figs. 3, 5, 6, 8). These mitochondria display a very electron-dense matrix in light-adaptation (Figs. 3, 5, 6). Smooth endoplasmic reticulum (SER) is widespread, while rough endoplasmic reticulum (RER) is very scarce (Figs. 2, 3, 4, 6). Polysomes are however widespread (Figs. 5, 6, 7, 8). Lysosome-like bodies and small (1.0 gm) lipid bodies were also present within these epithelial cells, with the lipid bodies being mainly located in the basal regions (Figs. 1, 3, 5, 6, 8). Melanosomes, which in this species are rod-shaped and measure up to 5 or 6 gm in length (Fig. 9), are in the light-adapted state predominantly located in the apical region of the cell bodies and within the apical processes (Figs. 1, 2, 4, 9). Within the apical processes they are aligned with the long axes of these processes and the photoreceptor cells (Figs. 2, 9). Myeloid bodies are a constant feature of these retinal epithelial cells, and in the light-adapted state are small and mostly located in the apical region of these cells (Figs. 1, 2). Phagosomes of shed outer segment material, while not abundant in these light-adapted specimens, are still present (Figs. 3, 5, 7). These phagosomes are often difficult to differentiate from the viable rod outer segments, which in light-adaptation also reach deep into the RPE cells (Figs. 1, 7, 8). As has been noted in most other teleost species, wandering phagocytes are a consistent feature of the retinal epithelial layer (Fig. 3). In these light-adapted specimens the wandering phagocytes were always located amongst the rod outer segments, and they usually contained phagosomes of outer segment material as well as melanosomes (Fig. 3). Bruch's membrane or complexus basalis in this species is typical of that noted in other teleosts in that it
is a trilaminate structure (Figs. 1, 5, 7, 8). It averages about 0.75 ~tm in thickness, and is composed of the basal lamina of the retinal epithelium and the: basal lamina of the choriocapillaris endothelium enclosing a thick layer of collagenous fibrils (Figs. 6, 7, 8). The choriocapillaris forms a single layer of large calibre capillaries immediately adjacent to the choroidal aspect of the complexus basalis (Figs. 1, 6, 7). The endothelium facing Bruch's membrane, while extremely thin, is only minimally fenestrated (Figs. 7, 8). Discussion
The retinal epithelium (RPE) of the velvet cichlid (Astronotus ocellatus) is basically similar to that described in other vertebrates, with modifications that seem to be specific to teleostean species (Rodieck 1973; NguyenLegros 1978; Kuwabara 1979; Braekevelt 1983, 1985a, 1989, 1991). As in all described species, the retinal epithelium in the velvet cichlid consists of a single layer of cells. In teleosts in general, the RPE cells are normally columnar, while in mammals reptiles and birds they tend to be cuboidal, and in marsupials and amphibians they are usually more squamous in shape (Braekevelt 1980a, 1982, 1984, 1988, 1990, 1991). The basal (scleral) aspect of the RPE of most vertebrate species is extensively infolded. This may indicate enhanced transport from the choriocapillaris (Dowling and Gibbons 1962; Bernstein and Hollenberg 1965; Bok 1985). Teleost species on the other hand normally show only minimal infolding in this region (Okuda 1962; Braekevelt 1980, 1982a). This is thought to be due to the presence of a choroid gland, which has been shown to maintain a high oxygen pressure, and presumably lessens the requirements of oxygen transport by the RPE in teleosts (Wittenberg and Wittenberg 1974). Apically (vitreally) retinal epithelial cells normally display numerous processes which serve to support the photoreceptor outer segment structurally (Bernstein 1961), as well as orienting them most effectively to the incoming light (Enoch 1979). These apical processes are also important in the internal adhesion required between the RPE and the photoreceptors (Zinn and BenjaminHenkind 1979). In addition, the intimate relationship between the apical processes of the RPE cells and the photoreceptors serves to facilitate the movement of materials between these cells (Dowling 1960; Steinberg and Wood 1974). The lateral borders of the retinal epithelial cells are normally relatively smooth, and are always joined by a series of tight junctions (Kuwabara 1979). It has been shown that these tight junctions (collectively forming Verhoeff's membrane) provide an effective barrier to the intercellular movement of materials, and hence form part of the blood-ocular barrier (Zinn and BenjaminHenkind 1979). Internally, RPE cells invariably display a large vesicular nucleus and an abundance of cell organelles (NguyenLegros 1978; Kuwabara 1979; Braekevelt 1988, 1990).
Fig. 1. Low power electron micrograph of the retinal epithelium of the velvet cichlid. The choriocapillaris (CC), rod outer segments (ROS) and retinal epithelial nuclei (iV) are indicated • 6,400 In all micrographs the scale bar equals 1 gm
Fig. 2. Electron micrograph of the apical region of the RPE layer. Melanosomes (M)~ myeloid bodies (MY) as well as cone inner (CIS) and outer (COS) segments are all labelled, x 8,600
Fig. 3. Electron micrograph to iUustrate a wandering phagocyte (WP), numerous mitochondria (Mi) and a phagosome (Ph) of outcr segment material. Bruch's membrane (B) is also indicated. • ]2,800
Fig. 4. Electron micrograph to indicate a b u n d a n t smooth cndoplasmic reticulum (SER), a rctinal epithelial cell nucleus (N) and a phagosome (Ph). x 13,200 Fig. 5. Electron micrograph of the R P E layer to illustrate mitochondria (M0, a cell junction (J) and a phagosome (Ph). x ]2,6~00
Fig. 6. Electron micrograph to indicate lipid droplets (L), mitochondria (Mi) and a rod outer segment (ROS). Bruch's membrane (B) is also indicated. • 13,500
Fig. 8. Electron micrograph to illustrate a lipid droplet (L), melanosomes (M) and a cell junction (3). Bruch's membrane (B) is also indicated, x 13,000
Fig. 7. Electron micrograph to indicate the lack of basal infoldings and relatively few fenestrations in the choriocapillaris (CC) endothelium. A cell junction (J) and a rod outer segment (ROS) are also labelled, x 15,800
Fig. 9. Electron micrograph to illustrate the rod-like shape of melanosomes (M) within the apical processes of the RPE. A rod photoreceptor (R) is also labelied, x 14,400
376 As has been noted in other species, smooth endoplasmic reticulum (SER) is abundant within RPE cells, while rough endoplasmic reticulum (RER) is not. The relative scarcity of R E R in these cells would indicate that little protein is being produced for extracellular destinations, while the abundance of polysomes indicates that intracellular protein requirements are normal. An abundance of SER is c o m m o n to cells involved in lipid biosynthesis (Enders 1962), and it is certainly well established that the RPE is heavily involved in the storage, transport and esterification of vitamin A (Berman 1979; Zinn and Benjamin-Henkind 1979). The presence of lysosomes in this epithelial layer is also to be expected, as one o f the most important functions of this layer is the phagocytosis and lysosomal degradation of outer segment discs (Young and Bok 1969; Young 1978). The relative scarcity of phagosomes within the RPE cells of this fish in the light-adapted state is probably due to the sampling time, as it is known that rods shed soon after lights are on and cones shed after lights are off (Young 1978). Myeloid bodies are a fairly constant feature within the R P E cells o f lower vertebrates which show retinomotor movements of the melanosomes (Nicol et al. 1975; Braekevelt 1980a, 1982). They normally appear as stacked SER membranes (Kuwabara 1979) while in avian species they may have ribosomes on their outer surface (Braekevelt 1984, 1990). They have been implicated as sites for storage of lipids prior to esterification (Yorke and Dickson 1984, 1985) and as the organelle that triggers photomechanical movements (Porter and Yamada 1960; Braekevelt 1982) but the function of myeloid bodies remains uncertain. The melanosomes within the RPE cells of the velvet cichlid are in the light-adapted state more numerous in the apical region of the cell body and within the apical processes, where they surround and isolate both rod and cone photoreceptor outer segments. While only lightadapted specimens were examined in this study, judging by the abundance and location of the melanosomes, retinomotor responses are thought to occur in the velvet cichlid, as is reported for all teleost species (Walls 1942). The wandering phagocytes noted amongst the RPE cells in the velvet cichlid are thought to be a normal, non-pathological feature of the teleost retina (Braekevelt 1980b, 1985b). As they are often seen to contain phagosomes of outer segment material they are probably involved in the phagocytosis and degradation of outer segment discs, as are the attached RPE ceils. Teleosts in general have very long outer segments, and retinomotor responses of the photoreceptors are normally rapid and extensive. These factors may cause an accelerated rate and/or volume of shed outer segment discs, and hence the wandering phagocytes may be supplementing the RPE cells in the phagocytosis of this material. O ' D a y and Young (1978) have indicated that in the goldfish these wandering phagocytes are much more numerous during the period of peak disc-shedding, which would tend to support their presumed role of aiding the attached RPE cells to remove outer segment debris. Bruch's membrane or complexus basalis in mammalian species is invariably pentalaminate, and displays a
central elastic layer (lamina densa), which is absent in teleost species, to give a trilaminate structure (Braekevelt 1980a, 1986, 1988). In avian species, while the lamina densa is present, it is normally very poorly represented (Braekevelt 1984, 1988, 1990). The significance (if any) of these phylogenetic variations within Bruch's membrane is at present unknown. The choriocapillaris in all described species is composed of a single layer of large calibre capillaries (Walls 1942; Rodieck 1973; Kuwabara 1979). With the exception of teleosts, the endothelium facing the complexus basalis is highly fenestrated, indicating the movement of large quantities of material across this endothelium (Bernstein and Hollenberg 1965). In teleosts the presence of a choroid gland which is important in the maintenance of a high oxygen pressure is probably responsible for the reduction of the number of fenestrations of the choriocapillaris endothelium, as well as the reduced number of basal infoldings, two features that are characteristic of teleost species (Braekevelt 1980a, 1985a).
Acknowledgements. The excellent technical assistance of D.M. Love and R. Simpson is gratefully acknowledged. This work was supported in part by funds from the Natural Sciences and Engineering Research Council (NSERC) and the Medial Research Council (MRC) of Canada. References
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