Cell Tiss. Res. 181,487-492 (1977) Cell and Tissue Research �9 by Springer-Verlag 1977

'Disk Shedding' in the Cone Outer Segments of the Teleost, Poecilia reticulata P.

Anwar Yacob and Yvette W. Kunz

Department of Zoology, University College, Dublin, Ireland

Summary. Electron microscopical observations show that the cones in the retina of the diurnal P o e c i l i a r e t i cu la ta shed their membranous outer seg- ment disks. This occurs at the side of the disk which is open to the extracellular space. Shedding is observed in single and twin cones and occurs at any level of the outer segment. The disks are not discarded in packages or as single disks, but are shed in small vesicular portions. This mode of 'disk shedding' may ex- plain why in cone outer segments radioactively labelled replacement protein is diffusely distributed.

Key words: Disk shedding - Cone outer segments - Teleost retina - P o e c i l i a

r e t i c u l a t a - Ultrastructure.

Introduction

Vertebrate visual cells - like other cells of the nervous system - once differentiated, do not divide any more. As a consequence, no replacement of damaged cells can take place. However, their photoreceptive outer segments are regenerated. Even under normal conditions, they are continually renewed by a constant supply of new proteins, as autoradiographic studies have shown (Young, 1967, 1968; Young and Droz, 1968). The rod outer segments (ROS) are renewed by discarding 'packages ' of disks at the apex and synthesizing new disks at the base. The renewal of cone outer segments (COS) is thought to involve neither the shedding of old disks nor the building of new ones, but some different, hitherto unknown, mecha- nism (Young, 1974). The following report, however, presents ultrastructural evidence of 'disk shedding' in the COS of the duplex retina of a viviparous teleost.

Material and Methods

The light adapted fish were decapitated, the eyes enucleated and fixed in 0.075 M buffered 2% glut- araldehyde, pH7.2. The tissue was postfixed in 1% osmium tetroxide, dehydrated in ethanol and

Send offprint requests to : Dr. Yvette Kunz-Ramsay, Department of Zoology, University College, Belfield, Dublin 4, Ireland

The authors wish to thank Dr. C. Wise of this Department for helpful discussions

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Fig. 1. Schematic drawing of a longitudinal section of a cone outer segment (O D showing the mem- hrarie shedding mechanism. A O S , accessory outer segment; CP, calycal process; I S , inner segment; MT, microtubules

Fig. 2. Longitudinally sectioned cone outer segment. Note that the detached vesicles (arrow) are expelled into the extracellular space, x 30,000. AOS, accessory outer segment; OS, outer segment; PE, pigment epithelium

Fig. 3. Transversely sectioned cone outer segment close to its scleral end showing the breakdown of disks into vesicles, x 25,000. dOS, accessory outer segment, with nine single microtubules; OS, outer segment

Fig. 4. Transverse section through cone outer segment (more vitreal than in Fig. 3). Note the shedding of the vesicles, x 25,000. AOS, accessory outer segment, with nine double microtubules; CP, calycal process; OS, outer segment

490 A. Yacob and Y.W. Kunz

embedded in Araldite. Ultrathin sections were stained with uranyl acetate, followed by lead citrate and examined with a Philips 201 C Electron microscope.

Results

The disks of photoreceptor outer segments arise as infoldings from the plasma membrane, as first proposed by Sj6strand (1961). In the ROS these infoldings separate entirely from the plasma membrane to form floating disks. In the COS, however, they remain continuous with the plasma membrane along the entire length of the outer segment. ('Cone saccules' would, therefore, be a more appropriate term than 'cone disks'.) As a result, the COS disks are enclosed, over most of their perimeter, by the plasma membrane, with only a small area open to the extracellular space (Cohen, 1972). This open area lies opposite the accessory outer segment (AOS) (Yacob et al., 1977).

In the retina of Poecilia the breakdown of COS membranes ('disk shedding') is repeatedly observed (Figs. 1, 2). It seems to be a gradual process, which begins with a marked swelling of the affected disk, accompanied by an accumulation of a diffuse, finely granular material of low electron-density. This is followed by the format ion of distended evaginations, which gradually lose their continuity with the rest of the disk and finally separate to form vesicles of various shapes and sizes. These vesicles are more abundant on the side opposite the AOS. Their presence results in the bending of the outer segment proper towards the AOS. Therefore, a cone undergoing 'disk shedding' shows slight tilts in the outer seg- ment axis (Fig. 2).

The detached vesicles migrate f rom the central part of the disk towards the periphery (open edge of the disk) and are finally liberated into the extracellular space between COS and pigment epithelial processes. The discharge of the vesi- cular material f rom the COS always takes place at the 'open disk-plasma mem- brane junct ion ' (Figs. 3, 4).

Disintegration and vesiculation of the disk membrane can take place at any level of the COS, as longitudinal and transverse sections demonstrate: a set of single microtubules in the AOS indicates a scleral level of a section (Fig. 3), whereas a set of double microtubules in the AOS and the presence of calycal processes (Fig. 4) are representative of a section at a vitreal level (Yacob et al., 1977).

Discus~on

A continuous renewal of mature ROS of various vertebrates was suggested by autoradiographic studies. Newly formed protein is concentrated at the base of the ROS and gradually moves sclerad, as a disk-shaped labelled band, until it reaches the apical end. Some of the renewal protein spreads diffusely through the ROS. In contrast, autoradiography of COS shows only a diffuse distribution of newly formed protein throughout the length of the COS (Young and Bok, 1969, 1970; Young, 1968, 1971a, b, c; 1974). The same results were obtained for the photoreceptors ofPoecilia reticulata, at a light microscopical level (Regan, 1977).

'Disk Shedding' in Cone Outer Segments 491

Electron microscopical observations confirm that ROS form new disks at their base and that their production is balanced by a shedding of old disks at the tip, thus maintaining a constant length of the ROS (Young and Bok, 1969; Young, 1971 a). A similar mechanism for COS is difficult to perceive from the autoradio- graphic studies and from a morphological point of view. A removal of 'disk packages' at the apical end would necessitate a continual rearrangement of the disk membranes to retain the conical shape of the COS. Nevertheless, Hogan et al. (1974) report apical shedding of cone disks in the human retina, but their findings are based on the study of only one eye. Conelike photoreceptors in the retina of tree squirrels have been shown to discard apical disks (Anderson and Fisher, 1975). The special mode of 'disk shedding' observed in Poecilia may explain how the conical shape of its COS is maintained.

'Disk shedding' in the COS may depend on exogenous and/or endogenous stimuli. It seems to be more pronounced after prolonged exposure to light (re- suits unpublished). It has been shown by La Vail (1976) that disk shedding in the ROS of the rat is initiated by light and follows a circadian rhythm. In frogs, 25~ of the rods shed their tips shortly after the onset of light; prolonged darkness decreases the amount of shedding (Basinger et al., 1976).

The broken-off tips of vertebrate ROS are engulfed by the pigment epithelium and enclosed in a cytoplasmic phagosome (Young, 1971 a). The COS in Poecilia are separated from the pigment epithelium by a comparatively large extracellular space. An intimate contact between COS and pigment epithelium is provided only by the AOS (Yacob et al., 1977). During this study, only one cone showed a lamellated body, resembling a package of COS disks, enclosed in the AOS. How- ever, the detachment of vesicular disk material from the open side of the COS was observed repeatedly in eyes of several specimens and in single as well as twin- cones.

The detachment of disk material in vesicular form and the fact that it occurs at any level of the COS may explain why in the COS labelled new proteins are diffusely distributed throughout the outer segment. The ultrastructural evidence of 'disk shedding' in Poecilia suggests that in this species the renewal of the COS takes place by the formation of new membranes, at multiple sites, to replace the shedded parts, and not simply by a replacement of molecules in the existing membranes.

Whether the disk material discarded by the COS into the extracellular space reaches the pigment epithelium to be phagocytized, or whether it has a different fate, can only be established by electron microscope autoradiography. Such ex- periments are currently in progress in this laboratory.

References

Anderson, D.H., Fisher, S.K. : Disc shedding in rodlike and conelike photoreceptors of three tree squirrels. Science 187, 953-954 (1975)

Basinger, S., Hoffman, R., Matthes, M.: Photoreceptor shedding is initiated by light in the frog retina. Science 194, 1074-1076 (1976)

Cohen, A.I. : Rods and cones. In: Handbook of sensory physiology, Vol. II/2. Physiology of photo- receptor organs, pp. 63-100 (M.G.F. Fuortes, ed.). Berlin-Heidelberg-New York: Springer 1972

492 A. Yacob and Y.W. Kunz

Hogan, M.J., Wood, I., Steinberg, R.H.: Phagocytosis by pigment epithelium of human retinal cones. Nature (Lond.) 252, 305-307 (1974)

La Vail, M.M.: Rod outer segment disk shedding in rat retina: Relationship to cyclic lighting. Science 194, 1071-1073 (1976)

Regan, C.M.: Purification and partial characterisation of teleost visual pigments. Ph.D. Thesis, National University of Ireland, Dublin (1977)

Sj6strand, F.S.: Electron microscopy of the retina. In: The structure of the eye, pp. 1-28 (G.K. Smelser, ed.). New York-London: Academic Press 1961

Yacob, A., Wise, C., Kunz, Y.W.: The accessory outer segment of rods and cones in the retina of the guppy, Poecilia reticulata P. (Teleostei). An electron microscopical study. Cell Tiss. Res. 177, 181-193 (1977)

Young, R.W.: The renewal of photoreceptor cell outer segments. J. Cell Biol. 33, 61-72 (1967) Young, R.W.: Passage of newly formed protein through the connecting cilium of retinal rods in the

frog. J. Ultrastruct. Res. 23, 462-473 (1968) Young, R.W.: Shedding of discs from rod outer segments in the rhesus monkey. J. Ultrastruct. Res.

34, 190-203 (1971 a) Young, R.W.: The renewal of rod and cone outer segments in the rhesus monkey. J. Cell Biol. 49,

303-318 (1971 b) Young, R.W.: An hypothesis to account for a basic distinction between rods and cones. Vision Res.

11, 1-5 (1971 c) Young, R.W.: Biogenesis and renewal of visual cell outer segment membranes. Exp. Eye Res. 18,

215-223 (1974) Young, R.W., Bok, D.: Participation of the retinal pigment epithelium in the rod outer segment

renewal process. J. Cell Biol. 42, 392-403 (1969) Young, R.W., Bok, D.: Autoradiographic studies on the metabolism of the retinal pigment epithelium.

Invest. Ophthalmol. 9, 524-536 (1970) Young, R.W., Droz, B.: The renewal of protein in retinal rods and cones. J. Cell Biol. 39, 169-184

(1968)

Accepted April 4, 1977