Ultrastructure of retinal pigment epithelium of the human fetus

© 1966 by Academic Press Inc.

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J. ULTRASTRUCTURERESEARCH16, 584--597 (1966)

U l t r a s t r u c t u r e of Retinal Pigment Epithelium of the H u m a n Fetus ~ A. S. BREATHNACHAND LUCILE M.-A. WYLLIE

Department of Anatomy, St. Mary's Hospital Medical School (University of London), London, England Received January 26, 1966 The ultrastructure of retinal pigment epithelium was studied in eyes of six human fetuses ranging in age from 8 to 14 weeks. General features of the cytoplasm and plasma membranes of cells at different stages are described, in comparison with the fully developed condition. The mode of development of the pigment granules of retinal cells is essentially similar to that occurring within the epidermal melanocytes. Small, membrane-limited vesicles, probably of Golgi origin, become larger and ovoid in shape, and acquire a system of concentric or folded inner membranes upon which melanin is deposited. The fine structure of the inner membranes of the granules and the manner in which they become melanized is likewise similar. Much information concerning the morphology and mode of production of melanin granules within the mammalian epidermal melanocyte has emerged from ultrastructural and biochemical Studies extending over the past decade (2, 3, 5, 11). There is little current support for the contention that melanin granules are specially modified mitochondria (6), and the most widely accepted view of their sequence of development is as follows: Tyrosinase is synthesized in ribosomes and transferred via the endoplasmic reticulum to the Golgi zone, where it accumulates in (or is incorporated within) small vesicles 0.05 # in diameter. These vesicles increase in size and acquire a system of internal membranes (either several concentrically arranged, or a single one wrapped in a spiral) along which the tyrosinase molecules become aligned in an ordered pattern. At this stage the organelles are known as premelanosomes, and with the onset of melanin deposition on the internal membranes they differentiate into melanosomes, i.e., partially melanized granules in which internal structure can still be discerned. Finally, with increasing melanin deposition, mature melanin granules, which are practically uniformly electron opaque, result. Two main steps are involved in the above-outlined scheme of melanin biosynthesis. Firstly, there is synthesis of z This study was supported by grants from the Medical Research Council and The Wellcome Trust.

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tyrosine-melanin by tyrosinase, a process which appears to be initiated in the endoplasmic reticulum (14) and continued within the premelansome and melanosome, and secondly, the assembly of the protein matrix of the inner membranes of these organelles with which the melanin combines, or upon which it is deposited. This latter process, it is thought, occurs exclusively within preformed, membrane-limited vesicles m o s t probably of Golgi origin (3, 11). The cells of the retinal pigment epithelium also produce melanin granules, but they differ from the epidermal melanocytes in some respects. The latter are derived developmentally from the neural crest, and constitute a reproductively self-maintaining :system of cells engaged in a continuous production of granules which they transfer to neighboring keratinocytes. The retinal cells stem from the outer wall of the optic cup and engage in melanogenesis for a limited period only, i.e., during fetal life and possibly for a short period post partum. They are also "continent" in the sense that they retain the formed melanin granules and do not pass them on to other cells. Considering these differences, it would not be altogether surprising if the morphology of the granules and certain features of their mode of production should likewise prove to be different in the two situations concerned. Moyer (9) has presented evidence from studies on the pigment epithelial ceils of the fetal mouse to support his view that this, in fact, is the case. We considered it of interest to determine whether the same applied to species other than the mouse, and in this report observations on the retinal pigment epithelium of the human fetus are presented. In addition to observations on the differentiation and morphology of the melanin granules, general features of the pigment cells at the particular fetal stages studied are described. MATERIALS AND METHODS Blocks of retinal pigment epithelium with underlying choroid were obtained from eyes of six human fetuses varying in age from 8 to 14 weeks. Following fixation for 2 hours in buffered osmium tetroxide at 4°C, the blocks were dehydrated in 70 %, 90 To, and absolute alcohols and then embedded in Araldite. Thin sections were cut on a Huxley ultramicrotome, stained with lead hydroxide on the grid (7), and examined in a Siemens Elmiskop I electron microscope. RESULTS

General arrangement of fetal pigment epithelium at 8-14 weeks Fig. 1 is a low-power micrograph of the pigment epithelium and choroid of a fetus aged 14 weeks. The pigment cells have large nuclei, and electron dense melanin granules are prominent in the cytoplasm. The basal plasma membranes of the cells rest u p o n a basement membrane (Fig. 2) which is separated from cellular elements of the vascular choroidal tissue by a narrow zone of loosely arranged collagen fibers. Isolated

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FIG. 1. Retinal pigment epithelium and subjacent choroid of 14-week fetus. Melanin granules (M)~ are present in the cytoplasm of the pigment cells. BM, basement membrane; E, erythrocyte within lumen of choriocapillary vessel; L, amorphous material possibly representing incomplete l a m i n a elastica choroidea, x 8500. FIG. 2. General aspect of a pigment cell of 14-week fetus. BM, basement membrane. Mature melanin granules (M), melanosomes (Me), and premelanosomes (Pm) are present in the cytoplasm, which: also contains mitoehondria (Mt), rough membranes (Rs), and smooth-surfaced vesicles of various size. N, nucleus; Tb, terminal bar. x 21,380.

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islands of amorphous material within this latter zone may represent an incomplete lamina elastica choroidea. Infolding of the basal plasma membrane which is such a prominent feature of cells of the fully developed epithelium (1) is not marked at 14 weeks, except at the basolateral angles, and is entirely absent at 8-10 weeks. The plane of lateral contact of adjacent cells at all stages examined, and particularly the later stages, was characterized by the presence of interdigitations of the plasma membrane, predominantly fine and short but occasionally in the form of larger finger-like processes. These latter were regularly circular in cross section, and their highly characteristic profiles were seen at various depths within the cytoplasm, some evidently penetrating to the juxtanuclear region (Fig. 3). Specialized contact zones, or terminal bars, were also present at intervals along the adjacent plasma membranes, particularly in the subapical zone (Fig. 2). The plasma membrane of the apical aspect of the cells was slightly undulant, but even in ~the later fetuses, there was little evidence of the epithelial fringes or processes which extend between the outer rod segments from this aspect of cells of the fully developed epithelium (1). Typical Golgi complexes of membranes and vesicles were seen in the juxtanuclear region of cells at all fetal stages examined (Figs. 4-6), and in general, the complexes at earlier stages presented an appearance of greater activity than those at later. Static appearances at 8-10 weeks (Fig. 6) in fact suggested that the Golgi complex was actively engaged in producing vesicles for wide dissemination throughout the cytoplasm, which was also characterized at this stage by networks of fine fibrils in peripheral areas, free ribosomal particles, moderate numbers of rough membranes and mitochondria, in addition to premelanosomes and melanosomes (see below). At later stages, e.g., 14 weeks, a filamentous network was less apparent, but all the organelles mentioned were more numerous, and some showed a tendency to be concentrated in particular regions of the cytoplasm, i.e., mitochondria in the basal zone, and rough membranes in the apical zone (Fig. 2). This distribution foreshadows the polar organization characteristic of cells of the fully developed epithelium (1). Premelanosomes and melanosomes Premelanosomes. In the outline of the sequence of development of melanin granules within the epidermal meianocyte given in the introductory section of this report, the fully differentiated premelanosome was described as an ovoid granule with an external limiting membrane and internal membranes exhibiting an ordered pattern (3).

FIG. 3. Sectional profile of finger-like process of an adjoining cell in the juxtanuclear region of a pigment cell of a 9-week fetus. N, nucleus, x 80,000. FIo. 4. Golgi membranes (Go) of pigment cell of 14-weekfetus. Pro, premelanosome. × 48,000. FIG. 5. Golgi membranes of pigment cell of 9-week fetus, x 63,000.

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Granules of identical appearance were found in cells of the retinal epithelium as the accompanying micrographs (Figs. 7-9) show. They were considerably larger than epidermal premelanosomes, the length varying between 0.9 # and 1.9 #, and the width between 0.3 ,u and 1.4 ,u. On section, the majority of granules exhibited inner membranes concentrically arranged (Fig. 7), but an occasional one presented a less regular internal structure of membranes folded in a more complicated fashion (Fig. 8). On longitudinal section the membranes appear as parallel lines when cut normally, and these exhibit an 80-90 A periodicity (Figs. 7 and 9) which is more evident in the early stages of melanin deposition (Fig. 12). A similar periodicity is evident on segments of membrane lying in the plane of sections above or below the axis of a granule (Figs. 9 and 13). The granules described above are mature premelanosomes within which differentiation of the protein matrix has been completed and which are about to undergo (or have already commenced) melanization. Also present in the cytoplasm were membrane-limited organelles of similar size with definite but less well-organized internal structure (Fig. 10) and vesicles of generally smaller diameter containing unorganized electron dense material (Figs. 6 and 11). These structures are identical in appearance, respectively, with the early premelanosomes and formative vesicles of epidermal melanocytes (3), and their presence in the retinal pigment cells strongly indicates that the origin and sequence of development of premelanosomes is essentially similar in the two situations concerned. In one respect, however, the cells in question may be said to differ. Whereas in epidermal melanocytes the early formative stages tend to be localized to the Golgi region, they may be encountered in all areas of the cytoplasm of the retinal cells. Melanosomes. These are granules undergoing progressive melanization, and representative stages are illustrated in Figs. 12-15. At early stages of melanin deposition an orthogonal arrangement of particles very similar to that described by Birbeck (3) is evident on segments of internal membrane lying in the plane of section (Fig. 13). The spacing of particles is likewise similar, being about 40-50 A across the granule and 80-90 A along its length. In longitudinal sections (Fig. 12) the inner membranes when cut normally exhibit in places the "helical thread" appearance which Birbeck (3) attributed to the deposition of melanin at alternate sites on either side of the FIG. 6. Cytoplasm of pigment cell of 8-weekfetus. Numerous small vesicleswhich appear to stem from the Golgi region (Go) are present. The organelles labeled Pro, are interpreted as later stages in the development of such vesicles into premelanosomes. Mt, mitochondria; N, nucleus, x 44,900. FIG. 7. Oblique section of premelanosome in pigment cell of 14-weekfetus. The internal membranes are concentrically arranged, and when sectioned normally, exhibit characteristic periodicity. L, limiting membrane. × 96,000. Fro. 8. Transverse section of premelanosome in pigment cell of 14-week fetus. The internal membranes are not concentrically arranged, being folded in a more complicated fashion, x 96,000.

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membrane. Other segments of sectioned membrane, however, presented the m o r e regular arrangement seen at A, Fig. 12. This appearance suggests that Birbeck's concept may require some revision in detail, but further examination of this matter must be postponed pending study of a large number of granules at high resolution. As melanization proceeds (Figs. 14 and 15) the inner membranes become progressively thicker and more electron dense, the intervals between them becoming narrower until the final conditon of a homogeneously electron dense-mature granule lacking any evident internal structure is reached. Where the majority of melanosomes are concerned, melanin deposition occurs uniformly throughout the granule. With some, however, it may be much further advanced in one part compared with another (Fig. 15). This condition has previously been observed by Moyer (9) in melanosomes of fetal mouse pigment epithelium.

DISCUSSION It is apparent from this study that premelanosomes and melanosomes within retinal pigment epithelial cells of the human fetus have essentially the same structure as similar organelles of the epidermal melanocytes (3). The evidence presented here further suggests an identical sequence of development from small membrane-limited vesicles which become larger and acquire a system of internal membranes exhibiting an ordered pattern. Whereas it is not possible to state with absolute certainty the source of the small vesicles, it seems likely that they stem from the Golgi region, whence similar precursor vesicles within the epidermal melanocytes are thought to be derived (3). Certainly, typical Golgi membranes are present within human retinal pigment epithelial cells, and, particularly at the earlier fetal stages examined, these appear to be actively engaged in the production of vesicles which become disseminated throughout the cytoplasm. The above observations and conclusions differ significantly from those reported by Moyer (9) following an examination of pigment epithelial cells of the fetal mouse. According to this author, "Golgi apparatus is absent from retinal pigment-producing cells...," and melanosomes are not derived from membrane-limited precursor vesicles. Further, the protein matrix of the granule does not consist of concentric or spirally FIG. 9. Longitudinal section of premelanosome in pigment cell of 14-week fetus. A considerable extent of one of the internal membranes is included in the section and exhibits characteristic periodicity. See also Fig. 13. L, limiting membrane. × 80,000. FIG. 10. Transverse section of premelanosome (Pro) at an earlier stage of development than those figured in Figs. 7-9. The internal membranes are becoming differentiated. From the apical region of pigment cell of 14-week fetus, x 80,000. FIG. 11. Area of basal cytoplasm of pigment cell of 14-week fetus. Early premelanosomes (Pro) of varying size are present. N, nucleus. × 56,000.

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FIG. 15. Transverse sections of melanosomes from pigment cell of 9-week fetus. The central granule is more heavily melanized in its central portion than at the periphery. The other granules exhibit varying degrees of disintegration of the inner membranes due to processing, thereby giving the impression of a fibrous internal structure. A, segments of disintegrated membrane presenting as fibers apparently lying free in the cytoplasm. × 40,000.

FIG. 12. Part of longitudinally sectioned melanosome at early stage of melanization. The "helical thread" appearance of normally sectioned membranes is evident toward the left. In the region A, a somewhat different appearance is observed, x 210,000. FI~. 13. Segment of internal membrane of melanosome at early stage of melanization. The orthogonal array of particles may be seen. x 300,000. FI~. 14. Transverse section of melanosome from pigment cell of 9-week fetus. The concentric arrangement of the internal membranes is clearly seen. × 80,000.

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arranged membranes, but of individual parallel fibers. These fibers are synthesized by ribosomes at many different sites within the cytoplasm and, by a process of aggregation and thickening, differentiate into melanosomes which secondarily acquire a limiting membrane. We would consider such a method of melanosome production to be fundamentally different to that proposed for cells of the present material, which is essentially similar to that described by previous workers for melanin-producing cells, both normal and abnormal in a variety of stituations in different species (2-5, 10, 12, 13). In all the instances referred to, the protein matrix of the granule takes shape within a membrane-limited vesicle, not free in the cytoplasm as described by Meyer (9). It is true that appearances similar to those illustrated by Meyer, and upon which his interpretation is based, were seen in some of our micrographs (see Fig. 15). However, we are satisfied that the occurrence in our material of what appeared to be granules with fibriUar inner structure, or aggregations of parallel fibers lying free in the cytoplasm, was due to disintegration during processing, or sectioning, of premelanosomes and melanosomes of membranous internal structure and with an outer limiting membrane. Insofar as one can judge from static morphological appearances, the process of melanin deposition on the membranous protein matrix of the premelanosomes of the present pigment epithelial cells is similar to that described by Birbeck (3) in epidermal melanocytes. An unusual feature, however, and one previously observed by Meyer (9) in the mouse, is the occurrence of melanosomes within which melanin deposition is irregular, being more advanced at a particular locus--usually the central region of the granule. We are not in a position at the moment to say what significance should be attached to this phenomenon. It may be that this type of granule is in some sense an abnormal or aborted melanosome. Alternatively, it could possibly represent a variant type of melanosome produced a t an earlier stage of development than the majority of those present. Examination of pigment epithelial cells at earlier fetal stages could provide more certain information on this point. In this connection it is of interest to note Koecke's (8) observation that melanocytes of the embryo duck produce successive crops of melanosemes of varying structure, though those of the final generation are of uniform morphology. REFERENCES 1. BAIRATI,A., JR., and ORZALESI,N., .J'. Ultrastruc. Res. 9, 484 (1963). 2. BARNICOT, N. A. and BIRBECK, M. S. C.. in MONTAGNA, W. and ELLIS, R. A. (Eds.),

The Biology of Hair Growth, p. 239. Academic Press, New York, 1958. 3. BIRBECK, M. S. C., Ann. N.Y. Acad. Sci. 100, 540 (1963). 4. BREATnNA¢~,A. S. and POYNTZ, S. V., J. Anat. 100, 549 (1966). 5. DROCHMANS, P., intern. Rev. Exptl. Pathol. 2, 357 (1963).

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6. DU BuY, H. G., SHOWACRE,J. L. and HESSELBACH,M. L., Ann. N.Y. Acad. Sci. 100, 569 (1963). 7. KARNOVSKY,M. J., J. Biophys. Biochem. Cytol. 11, 729 (1961). 8. KOECKE, H. O., personal communication, 1965. 9. MOYER,F. H., Ann. N.Y. Acad. Sci. 100, 584 (1963). 10. RAPPAVORT,H., NAKAI, T. and SWIFT, H., J. Cell Biol. 16, 171 (1963). 11. SEre, M., SmMAO, K., BmBECK, M. S. C. and FITZVATRICK,T. B., in RILEY, V. and FORTNER, J. G. (Eds.), The Pigment Cell, Ann. N.Y. Acad. Sci. 100, 497 (1963). 12. WELLINGS,S. R. and SIEGEL, B. V., Ann. N.Y. Acad. Sci. 100, 548 (1963). 13. WELLINGS,S. R. and SIEGEL, B. V., J. Natl. Cancer Inst. 24, 437 (1960). 14. ZELICKSON,A. S., HIRSCH, H. B. and HARTMANN,J. F., 3". Invest. Dermatol. 43, 327 (1964).