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Cytochemical localization of tyrosinase activity in pigmented epithelial cells of Rana pipiens and Xenopus laevis larvae
1972 by Academic Press, Inc.
S. ULTRASTRUCTURE RESEARCH 39, 397-410 (1972)
397
Cytochemical Localization of Tyrosinase Activity in Pigmented Epithelial Cells of Rana pipiens and Xenopus laevis Larvae JOHN J. EPPIG, JR. z a n d JAMES N . DUMONT
Biology Division, Oak Ridge National Laboratory, 2 Oak Ridge, Tennessee 37830 Received September, 8, 1971, and in revised form December 15, 1971 Tyrosinase has been localized by the DOPA reaction in the pigmented epithelium of larval Rana pipiens and Xenopus laevis. Reaction product is found in the distal cisternae (active face) of the Golgi apparatus and in a continuous system of smooth-surfaced channels which communicate with both premelanosomes and developing complex melanosomes. In addition, reaction product is found in tubules and cisternae, which sometimes appear as an intricately folded system connecting with premelanosomes in several places and interconnecting neighboring premelanosomes. The evidence suggests that tyrosinase is active in DOPA-positive channels but not in early premelanosomes. Thus, melanogenesis involves the dynamic processes of tyrosinase condensation in the Golgi apparatus, with melanin synthesis beginning in the channels and melanin deposition occurring in premelanosomes. As the premelanosomes mature, tyrosinase is transferred to them; thus progressively more melanin synthesis may occur in the intermediate or late premelanosomes.
The melanosomes of amphibians (5, 6) and of higher vertebrates (2-4, 13, 16, 20, 26) are formed by the deposition of melanin on a membrane-bound fibrillar matrix called the premelanosome. According to current terminology, the term premelanosome is applied to all stages in the formation of melanosomes and the term melanosome is reserved for the "fully pigmented melanin-containing organelle" (8). We shall use the term early premelanosome to indicate the membrane-bound fibrillar structure prior to any melanin deposition, and the terms intermediate and late premelanosome are used to refer to stages of melanosome development in which progressively more pigment is deposited upon this fibrillar matrix. The initial steps in the biosynthesis of melanin involve the conversion of L-tyrosine to L-dihydroxyphenylalanine (DOPA), followed by the conversion of D O P A to DOPA-quinone. It is generally z University of Tennessee Postdoctoral Investigator under contract No. 3322 with the Biology Division, Oak Ridge National Laboratory. Operated by the Union Carbide Corporation for the U.S. Atomic Energy Commission. 26 -- 721831 J . Ultrastructure Besearch
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considered that both these reactions are catalyzed by the enzyme tyrosinase; however, Okun et al. (19) suggest that peroxidase may also play a role in the biosynthesis of melanin. Tyrosinase can be localized cytochemically by incubating fixed tissue in a medium containing DOPA. The reaction product, probably melanin, is identified in the electron microscope by its high density. Many studies using this cytochemical method on a variety of melanogenic cell types have been conducted (9, 13, 15, 18, 25). Some authors (13, 15, 25) suggest that early premelanosomes and their matrices are formed from dilations of the endoplasmic reticulum and that tyrosinase is transferred to them from the Golgi apparatus either by Golgi vesicles or through tubules directly connecting the Golgi apparatus and the premelanosomes. On the other hand, earlier biochemical and ultrastructural studies led to the proposal that premelanosomes themselves form directly from smooth-surfaced, tyrosinase-containing vesicles derived from the Golgi complex (3, 23, 27). According to these studies, the tyrosinase becomes activated in the premelanosomes and melanin is deposited within them. The experiments reported here were undertaken to extend the observations on the cytological localization of tyrosinase to the pigmented epithelium of larval amphibians. These cells are of special interest since they contain not only melanosomes which develop from typical fibrillar premelanosomes, but also complex melanosomes which are formed by the deposition of new melanin around melanosomes derived from the oocyte (5, 7). Thus, in this context, the oocyte melanosomes act as premelanosomes which can accept further melanin deposition. It is difficult to reconcile this with the theory that premelanosomes form directly from smooth-surfaced, tyrosinase-containing vesicles derived from the Golgi apparatus because it would necessitate the existence of dual mechanisms of tyrosinase transfer. A simpler mechanism would provide for the direct transfer of tyrosinase from the protein synthesizing and condensing compartments of the cell to both fibrillar premelanosomes which form from the endoplasmic reticulum and to the developing complex melanosomes which use oocyte melanosomes as their base for melanin deposition. The experiments reported here demonstrate that such a direct means of tyrosinase transfer exists between the Golgi complex and fibrillar premelanosomes and developing complex melanosomes in the pigmented epithelium of larval eyes. FIG. 1. Electron micrographs showing the localization of DOPA reaction in the pigmented epithelium of Rana pipiens larvae. (a) Golgi complexes are oriented with DOPA-positive cisternae toward (G1) and away (G2) from the apical, i.e., melanosome, region of the cell. DOPA-positive cisternae and tubules (L) loop from surface of late premelanosomes; some late premelanosomes have undulating or scalloped borders. Sections of DOPA-positive tubules (T) are present in the cytoplasm. N, Nucleus. (b) Early premelanosomes (P) do not contain DOPA-reaction product. This slightly oblique section illustrates DOPA-positive fenestrated cisternae (arrow) of Golgi complex. (a) x 68 000; (b) x 50 000.
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Adult Rana pipiens were purchased from J. R. Schettle Biologicals, Stillwater, Minnesota, and Xenopus laevis from Jay Cook, Cockeysville, Maryland. Some larvae were reared in spring water and others in spring water containing 0.005% or 0.0075% phenylthiourea (PTU) to prevent melanin synthesis (5, 6). Stage 22 to 24 R. pipiens larvae (24) and stage 33/34 X. laevis larvae (17) were used for the study. R. pipiens larvae were decapitated, and the eyes were excised while the heads were immersed in cold 3 % glutaraldehyde in 0.1 M phosphate buffer at pH 7.4. When all eyes were collected, fresh fixative was substituted for a total fixation time of 2 hours. Since it is difficult to excise the eyes of Xenopus without injuring them, the larvae were decapitated and the heads fixed for 1 hour. The heads were then cut in half sagitally, and fresh fixative was added for another hour. All tissues were washed for 15 minutes in 0.1 M phosphate buffer containing 6.8 % sucrose and incubated in a medium containing 0.1% (w/v) L-DOPA (Sigma Chemical Company) in 0.1 M cacodylate buffer with 6.8 % sucrose at pH 7.4 for 2.5 hours at 25.5°C. Rodriguez and McGavran (22) suggest that a cacodylate buffer be used in the DOPA medium since it inhibits DOPA autoxidation. The eyes from R. pipiens larvae reared in 0.005 % PTU were fixed as indicated above. They were, however, washed overnight in the DOPA medium at 4°C in an attempt to remove the PTU. The medium was then changed, and the eyes were incubated in three changes of DOPA medium for an additional 6.5 hours at 25.5°C. Control eyes for the DOPA reaction were incubated without DOPA or in a DOPA medium containing 0.01% PTU as an inhibitor. Since it has been suggested (19) that peroxidase is involved in the initial steps of melanin synthesis in some systems, some eyes were also incubated in the diaminobenzidine medium of Graham and Karnovsky, as described by Okun et al. (19). To test this implication, some eyes were treated with 0.1% catalase in 0.t M cacodylate buffer containing 6.8 % sucrose at 25.5°C for 30 minutes and then incubated in DOPA medium also containing catalase. After the various incubations, all tissues were washed in two changes of cold 0.1 M phosphate buffer containing 6.8% sucrose for a total of 15 minutes and postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer, also containing 6.8% sucrose. All tissues were embedded in Epon and sectioned with a Porter-Blum MT-2 ultramicrotome, stained with uranyl acetate followed by lead citrate, and examined with a Hitachi HU-11E electron microscope operated at 75 kV. To obtain three-dimensional perspective, serial sections were analyzed by tracings from electron micrographs. RESULTS
D OPA reaction in normal pigmented epithelial cells The m o r p h o l o g y of the pigmented epithelium of n o r m a l a n d P T U - t r e a t e d R.
pipiens a n d X. Laevis larvae has already been described (5, 7). The larvae used for FIG. 2. Electron micrograph illustrating the intricate system of DOPA-positive tubules (7") which link clusters of late premelanosomes in Rana pipiens, x 75 000. Fro. 3. The interconnecting DOPA-positive tubules (T) are present but less extensive in Xenopus laevis. Note the scalloped appearance of the boundary of the late premelanosomes, x 75 000.
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the present experiments were younger than those studied previously. The only differences noted were the absence of myeloid bodies and the more frequent appearance of Golgi complexes and early premelanosomes. When the tissue has been incubated in the DOPA medium, electron-dense reaction product is observed in the cisternae of the distal or active face Of the Golgi apparatus and occasionally in coated Golgi vesicles (Fig. la). Although the pigmented epithelial cells are polarized, so that the melanosomes are primarily in the apical cytoplasm, the active face of the Golgi apparatus may lie el[her toward or away from the apical surface of the cell (Fig. 1 a). No reaction product is observed in early premelanosomes (Fig. 1 b); but it is difficult to ascertain whether electron dense material in intermediate and late premelanosomes is reaction product or naturally deposited melanin. The peripheries of some premelanosomes in eyes incubated in DOPA medium often have a scalloped appearance (Figs. 1 a and 3) which is absent in the controls, suggesting that DOPAreaction product is deposited in this region. According to Fitzpatrick et al. (8), melanosomes are complete organelles containing little or no tyrosinase activity. Therefore, the scalloped structures, although relatively homogeneous in electron density, are presumably late premelanosomes. The melanosomes of more mature melanocytes are not scalloped after incubation in DOPA medium. DOPA-reaction product is also present in an anastomosing system of tubules which interconnect premelanosomes and developing complex melanosomes (Figs. 2 and 3). In addition, reaction product was observed in continuities directly linking the Golgi apparatus with both premelanosomes and developing complex melanosomes (Figs. 4 and 5). Occasionally, late premelanosomes are encompassed in an intricate DOPApositive network which appears in the form of loops in individual micrographs. However, serial sections show that these looped structures are continuous through 5-8 sections and must, therefore, extend at least 300 nm (Fig. 6). These broad structures are therefore considered to be intricately folded cisternal structures which merge with the premelanosome membrane in several places and are continuous with the DOPA-positive tubules linking other premelanosomes.
D OPA reaction in pigmented epithelial cells of larvae reared in phenylthiourea (PTU) The DOPA reaction, as well as melanin synthesis can be inhibited by PTU, presumably because P T U binds with copper ions which are essential for tyrosinase FIG. 4. Four serial sections (total thickness -0.12 p) illustrating the relationship of developing complex melanosomes (C) and elongated late premelanosomes (E) to cisternae of the Golgi complex (6) in Rana pipiens. The arrow (Fig. 4c) suggests a direct continuity, x 36 000.
i 1
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activity (12). However, to ascertain the localization of tyrosinase in the pigmented epithelium of PTU-treated larvae, an attempt was made to remove the P T U from the fixed tissues and hopefully reactivate the tyrosinase. This was done by washing the tissue overnight in DOPA medium at 4°C and reincubating in fresh D O P A medium at 25.5°C. Although the extent of PTU removal is uncertain, the washed pigmented epithelial cells of PTU-treated larvae nevertheless contained reaction product in the Golgi apparatus and associated smooth membrane systems (Fig. 7), whereas unwashed cells did not contain any reaction product. In the washed cells, little reaction product was found in premelanosomes which would normally develop to form elongate melanosomes, nor was reaction product associated with oocyte melanosomes which would normally become complex melanosomes. Controls
No DOPA-reaction product was found in normal eyes incubated in medium-con taining P T U or in substrate-free medium. In addition, non-pigment-producing cells of eyes incubated in DOPA medium contained no reaction product. No peroxidase reaction product was observed in tissues incubated in diaminobenzidine; and catalase in the DOPA medium did not affect the accumulation of DOPA-reaction product in the cells. These results suggest that peroxidase plays no role in the formation[of the DOPA reaction product observed in these amphibian pigmented epithelial cells. DISCUSSION Our results confirm that tyrosinase is localized in elements of the Golgi apparatus and that early premelanosomes contain little or no demonstrable tyrosinase. Both premelanosomes and developing complex melanosomes are linked to the Golgi apparatus by DOPA-positive, smooth-surfaced channels. In addition, both are interconnected by an anastomosing network of DOPA-positive tubules. Some premelanosomes are encompassed by intricately folded, DOPA-positive cisternal elements, which merge with the premelanosome membrane and interconnect with tubules joining other developing melanosomes. Our interpretation of some possible forms and associations of the network is illustrated in Fig. 8. Since this network appears to be greatly reduced in older cells, it may be a transient structure. However, it is difficult to ascertain whether the system itself is transient or merely ceases to be DOPA positive. FIG. 5. Adjacent serial sections showing continuity (arrows) of late premelanosome and cisternae of Golgi complex (G) in Rana pipiens, x 44 000. Ft6. 6. Serial sections (total thickness = 0.3 #) demonstrate in Rana pipiens the intricately folded system of DOPA-positive cisternae which encompass some late premelanosomes. Note connections with the late premelanosomes (arrows). x 36 000.
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The presence of DOPA-reaction product in a particular location in a fixed cell does not confirm that tyrosinase is active at that site in the living cell. For example, it may be that intracellular tyrosinase inhibitors are unbound, washed out, or inactivated during fixation, thus activating the tyrosinase. On the basis of his ultrastructural and biochemical studies, Seiji (23) suggested that although tyrosinase can be demonstrated in membrane as well as premelanosome fractions isolated from melanomas, actual melanin synthesis occurs only in premelanosomes which have received their quantum of tyrosinase before separation from the Golgi appartaus. Membrane digestion studies by Seiji (23) suggest that tyrosinase may be so tightly bound to isolated smooth-surfaced membranes that it is actually an integral part of the membrane. Yet, those smooth-surfaced membranes did not contain significant amounts of radioactivity after animals were injected with 14C-DOPA; only the fractions which contained premelanosomes were significantly radioactive. It seems possible, however, that melanin precursors not bound to the membranes may have been washed out during the membrane isolation procedure. This would account for the interpretation that melanin synthesis occurs only in premelanosomes. Complex melanosomes in the pigmented epithelium of larval amphibians seem to provide an additional complication for Seiji's hypothesis that premelanosomes form directly from smooth-surfaced, tyrosinase-containing vesicles derived from the Golgi apparatus. The complex melanosomes are formed by the deposition of new melanin around oocyte melanosomes which in this environment may be regarded as premelanosomes. Thus, in this case these premelanosomes preexist in the cell and are clearly not formed from tyrosinase-containing Golgi vesicles. Our evidence shows that developing complex melanosomes, as well as premelanosomes, are connected directly to the Golgi complex by smooth-surfaced, DOPA-positive channels-channels through which enzyme is transported. An experiment was designed to provide further insight into the question of where tyrosinase is active in the living cell. This experiment utilized the eyes of larvae reared in P T U to inhibit melanin synthesis. Premelanosomes are formed in the pigmented epithelium of such PTU-treated larvae, but no pigment is deposited on them (5). In PTU-treated larvae these premelanosomes appear as early premelanosomes, but under normal circumstances they would have become melanized. We proposed two possibilities for the localization of DOPA-reaction product in cells from which all or some of the PTU was removed by washing prior to incubation in DOPA medium. The two possibilities are: (a) If the tyrosinase passes through the channel linking the
FIG. 7. An electron micrograph of pigmented epithelium from Rana pipiens larvae reared in PTU. PTU was washed out of tissue prior to incubation in DOPA medium. The reaction product is localized in tubules and cisternae but not in premelanosomes (P). x 10 900.
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Golgi apparatus and premelanosomes to become active only at this site in the living cell, then it should accumulate there. In this case, DOPA reaction product would be dense in the premelanosomes after removal of PTU. (b) If tyrosinase is not active primarily in the premelanosomes then enzyme activity should be localized in the linking channels of the PTU-treated cells. The latter proved to be the case. Most of the reaction product was localized in the linking membrane system rather than in the premelanosomes. This experiment suggests that tyrosinase activity and, therefore, melanin synthesis at least begin in the DOPA-positive channels which link the Golgi apparatus with premelanosomes. Some evidence indicates that at high concentrations PTU slightly inhibits protein synthesis (11). If the net synthesis of tyrosinase is affected by the concentration of PTU used in rearing the larvae, it could present some serious problems in the interpretation of our experiment. It has been shown, however, that concentrations of PTU greater than that used in our experiments do not inhibit the synthesis of tyrosinase in ascidian embryos (28). In addition, it is unlikely that tyrosinase synthesis and intracellular transport are seriously affected by our procedure, since the larvae as a whole develop normally (except for pigmentation) in PTU. Autoradiographic studies tracing the incorporation of 3H-DOPA into melanoma melanocytes led Zelickson et al. (10, 29) to suggest that melanin or its immediate insoluble precursors are present in the smooth endoplasmic reticulum. They propose that tyrosinase is active within the membrane system as well as in the premelanosomes. Our results support this hypothesis by suggesting that in the living cell tyrosinase is active in the DOPA-positive channels which link the Golgi complex and premelanosomes. If this is true, melanogenesis involves the dynamic process of enzyme condensation in the Golgi apparatus, biosynthesis of melanin beginning in the DOPA-positive channels and deposition occurring in premelanosomes. The relative proportion of melanin synthesis in the various cellular compartments, however, is still open to question. It is possible that as the premelanosomes mature, tyrosinase is transferred to them; thus, progressively more melanin synthesis may occur in the intermediate or late premelanosomes. Apparently some melanogenesis occurs in these premelanosomes, as evidenced by the scalloped appearance of their peripheries after incubation in DOPA medium. The transfer of material to the premelanosome via channels from the Golgi complex is a variance from the usual mode of operation proposed for the Golgi apparatus. In most systems it apparently functions by condensing and transferring material received from rough endoplasmic reticulum (1). The condensed material is sequestered in the cytoplasm by the pinching off or blebbing of vesicles from the Golgi cisternae. Some of these vesicles may then fuse to form larger vesicles or granules. However, Reddy and Svoboda (21) have demonstrated "numerous continuities (1) between
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FIG. 8. Illustration of some of the possible intricate configurations and associations of the DOPA-
positive membrane system. These smooth-surfaced membranes may be in the form of tubules (T) or cisternae (C). Some portions have been "cut away" (arrows) to illustrate how some of the "loops" and connections, as seen in electron micrographs, may occur in three-dimensional perspective. P1 is a late premelanosome with its bounding membrane intact, while P2 shows a naked melanosome with the associated networks of DOPA-positive cisternae cut away. A complex melanosome (CM) is shown conneted to P2 and the active face of the Golgi apparatus (G) by DOPA-positive tubules.
the limiting membranes of microbodies and rough and smooth endoplasmic reticulum and (2) between two adjacent microbodies or more" in liver cells of acatalasemic rats treated with chlorophenoxyisobutyrate (CPIB). Administration of CPIB induces a rapid proliferation of microbodies in the liver. Reddy and Svoboda suggest that the microbody connections may allow an interchange of material relative to rapidly altering protein pools. In like manner, the interconnecting network of D O P A positive tubules and cisternae linking developing premelanosomes may be established to facilitate the rapid maturation of a large number of premelanosomes during differentiation in the pigmented epithelium. In this connection, our results as well as those of Maul (13) indicate that connections between smooth endoplasmic reticulum and the premelanosomes were seen less frequently as melanization of premelano-
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somes neared completion. It seems probable, therefore, that such interconnecting membrane systems are broken down after their role in melanogenesis is completed. The authors wish to thank Gay Ann Eppig for the preparation of Fig. 8.
REFERENCES 1. 2. 3. 4.
BEAMS,H. W. and KESSEL, R. G., Int. Rev. CytoL 23, 209 (1968). BREATHNACH,A. S. and POYNTZ, S. V., J. Anat. 100, 549 (1966). BREATHNACH,A. S. and WYLIE, L. M., J. Ultrastruct. Res. 16, 584 (1966). DROCHMANS,P., Advances in Biology of the Skin, The Pigmentary System, Vol. VII, p. 169. Pergamon Press, London, 1967. 5. EPVlG, J. J., JR., Z. Zellforsch. Mikrosk. Anat. 103, 238 (1970). 6. - J. Embryol. Exp. Morphol. 24, 447 (1970). 7. - J. Exp. Zool. 175, 467 (1970). 8. FITZPATRICK,T. B., QUEVEDO, W. C., LEVENE, A. L., McGOVERN, V. J., MISHIMA, Y. and OETTLE,A. G., Science, 152, 88 (1966). 9. GUTTES,E. and BRANDT, S. M., J. Histochem. Cytochem. 9, 457 (1961). 10. HIRSCH, H. H., ZELICKSON,A. S. and HARTMANN,J. F., Z. Zellforsch. Mikrosk. Anat. 65, 409 (1965). 11. KITANO,Y. and Htr, F., Exp. Cell. Res. 64, 83 (1971). 12. LERNER,A. B., CALKINS,E. and SUMMERSON,W. H., J. Biol. Chem. 187, 793 (1950). 13. MAUL, G. G., J. Ultrastruct. Rex. 26, 163 (1969). 14. MAUL, G. G. and ROMSDAHL,M. M., Cancer Res. 30, 2782 (1970). 15. MAt~L, G. G. and BRt~MBAU~H,J. A., J. Cell Biol. 48, 41 (1971). 16. MOYER,F. H., Amer. ZooL 6, 43 (1966). 17. NIEUWKOOP, P. D. and FABER, J., Normal Table of Xenopus laevis (Daudin). North Holland Publ., Amsterdam, 1956. 18. NOVtKOEE,A., ALBALA,A. and BIEMVlCA,L., J. Histochem. Cytochem. 16, 299 (1968). 19. OKUN, M. R., EDELSTEIN,L. M., OR, N., HAMADA,G., DONNELLAN,B. and LEVER,W. F. Histochemie 23, 295 (1970). 20. RAPPAPORT,H., NAKAI, T. and SWIFT, H., J. Cell. Biol. 16, 171 (1963). 21. REDDY, J. and SVOBODA,D., Lab. Invest. 24, 74 (1971). 22. RODRIGUEZ,H. A. and McGAVRAN, M. H., Amer. J. Clin. Pathol. 52, 219 (1969). 23. SEre, M., Advances in Biology of the Skin, The Pigmentary System, Vol. VIII, p. 189. Pergamon Press, London, 1967. 24. SHUMWAY,W., Anat. Rec. 78, 139 (1940). 25. STANKA,P., Mikroskopie 26, 169 (1970). 26. - Z. Zellforsch. Mikrosk. Anat. 112, 120 (1971). 27. WELLINGS,S. R. and SEIGEL,B. V., J. Ultrastruct. Res. 3, 147 (1963). 28. WHITTAKER,J. R., Develop. Biol. 14, 1 (1966). 29. ZELICKSON,A. S., HIRSCH, H. M. and HARTMANN,J. F., J. lnvest. DermatoI. 45, 458 (1965).
S. ULTRASTRUCTURE RESEARCH 39, 397-410 (1972)
397
Cytochemical Localization of Tyrosinase Activity in Pigmented Epithelial Cells of Rana pipiens and Xenopus laevis Larvae JOHN J. EPPIG, JR. z a n d JAMES N . DUMONT
Biology Division, Oak Ridge National Laboratory, 2 Oak Ridge, Tennessee 37830 Received September, 8, 1971, and in revised form December 15, 1971 Tyrosinase has been localized by the DOPA reaction in the pigmented epithelium of larval Rana pipiens and Xenopus laevis. Reaction product is found in the distal cisternae (active face) of the Golgi apparatus and in a continuous system of smooth-surfaced channels which communicate with both premelanosomes and developing complex melanosomes. In addition, reaction product is found in tubules and cisternae, which sometimes appear as an intricately folded system connecting with premelanosomes in several places and interconnecting neighboring premelanosomes. The evidence suggests that tyrosinase is active in DOPA-positive channels but not in early premelanosomes. Thus, melanogenesis involves the dynamic processes of tyrosinase condensation in the Golgi apparatus, with melanin synthesis beginning in the channels and melanin deposition occurring in premelanosomes. As the premelanosomes mature, tyrosinase is transferred to them; thus progressively more melanin synthesis may occur in the intermediate or late premelanosomes.
The melanosomes of amphibians (5, 6) and of higher vertebrates (2-4, 13, 16, 20, 26) are formed by the deposition of melanin on a membrane-bound fibrillar matrix called the premelanosome. According to current terminology, the term premelanosome is applied to all stages in the formation of melanosomes and the term melanosome is reserved for the "fully pigmented melanin-containing organelle" (8). We shall use the term early premelanosome to indicate the membrane-bound fibrillar structure prior to any melanin deposition, and the terms intermediate and late premelanosome are used to refer to stages of melanosome development in which progressively more pigment is deposited upon this fibrillar matrix. The initial steps in the biosynthesis of melanin involve the conversion of L-tyrosine to L-dihydroxyphenylalanine (DOPA), followed by the conversion of D O P A to DOPA-quinone. It is generally z University of Tennessee Postdoctoral Investigator under contract No. 3322 with the Biology Division, Oak Ridge National Laboratory. Operated by the Union Carbide Corporation for the U.S. Atomic Energy Commission. 26 -- 721831 J . Ultrastructure Besearch
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considered that both these reactions are catalyzed by the enzyme tyrosinase; however, Okun et al. (19) suggest that peroxidase may also play a role in the biosynthesis of melanin. Tyrosinase can be localized cytochemically by incubating fixed tissue in a medium containing DOPA. The reaction product, probably melanin, is identified in the electron microscope by its high density. Many studies using this cytochemical method on a variety of melanogenic cell types have been conducted (9, 13, 15, 18, 25). Some authors (13, 15, 25) suggest that early premelanosomes and their matrices are formed from dilations of the endoplasmic reticulum and that tyrosinase is transferred to them from the Golgi apparatus either by Golgi vesicles or through tubules directly connecting the Golgi apparatus and the premelanosomes. On the other hand, earlier biochemical and ultrastructural studies led to the proposal that premelanosomes themselves form directly from smooth-surfaced, tyrosinase-containing vesicles derived from the Golgi complex (3, 23, 27). According to these studies, the tyrosinase becomes activated in the premelanosomes and melanin is deposited within them. The experiments reported here were undertaken to extend the observations on the cytological localization of tyrosinase to the pigmented epithelium of larval amphibians. These cells are of special interest since they contain not only melanosomes which develop from typical fibrillar premelanosomes, but also complex melanosomes which are formed by the deposition of new melanin around melanosomes derived from the oocyte (5, 7). Thus, in this context, the oocyte melanosomes act as premelanosomes which can accept further melanin deposition. It is difficult to reconcile this with the theory that premelanosomes form directly from smooth-surfaced, tyrosinase-containing vesicles derived from the Golgi apparatus because it would necessitate the existence of dual mechanisms of tyrosinase transfer. A simpler mechanism would provide for the direct transfer of tyrosinase from the protein synthesizing and condensing compartments of the cell to both fibrillar premelanosomes which form from the endoplasmic reticulum and to the developing complex melanosomes which use oocyte melanosomes as their base for melanin deposition. The experiments reported here demonstrate that such a direct means of tyrosinase transfer exists between the Golgi complex and fibrillar premelanosomes and developing complex melanosomes in the pigmented epithelium of larval eyes. FIG. 1. Electron micrographs showing the localization of DOPA reaction in the pigmented epithelium of Rana pipiens larvae. (a) Golgi complexes are oriented with DOPA-positive cisternae toward (G1) and away (G2) from the apical, i.e., melanosome, region of the cell. DOPA-positive cisternae and tubules (L) loop from surface of late premelanosomes; some late premelanosomes have undulating or scalloped borders. Sections of DOPA-positive tubules (T) are present in the cytoplasm. N, Nucleus. (b) Early premelanosomes (P) do not contain DOPA-reaction product. This slightly oblique section illustrates DOPA-positive fenestrated cisternae (arrow) of Golgi complex. (a) x 68 000; (b) x 50 000.
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EPPIG, JR AND DUMONT MATERIALS A N D METHODS
Adult Rana pipiens were purchased from J. R. Schettle Biologicals, Stillwater, Minnesota, and Xenopus laevis from Jay Cook, Cockeysville, Maryland. Some larvae were reared in spring water and others in spring water containing 0.005% or 0.0075% phenylthiourea (PTU) to prevent melanin synthesis (5, 6). Stage 22 to 24 R. pipiens larvae (24) and stage 33/34 X. laevis larvae (17) were used for the study. R. pipiens larvae were decapitated, and the eyes were excised while the heads were immersed in cold 3 % glutaraldehyde in 0.1 M phosphate buffer at pH 7.4. When all eyes were collected, fresh fixative was substituted for a total fixation time of 2 hours. Since it is difficult to excise the eyes of Xenopus without injuring them, the larvae were decapitated and the heads fixed for 1 hour. The heads were then cut in half sagitally, and fresh fixative was added for another hour. All tissues were washed for 15 minutes in 0.1 M phosphate buffer containing 6.8 % sucrose and incubated in a medium containing 0.1% (w/v) L-DOPA (Sigma Chemical Company) in 0.1 M cacodylate buffer with 6.8 % sucrose at pH 7.4 for 2.5 hours at 25.5°C. Rodriguez and McGavran (22) suggest that a cacodylate buffer be used in the DOPA medium since it inhibits DOPA autoxidation. The eyes from R. pipiens larvae reared in 0.005 % PTU were fixed as indicated above. They were, however, washed overnight in the DOPA medium at 4°C in an attempt to remove the PTU. The medium was then changed, and the eyes were incubated in three changes of DOPA medium for an additional 6.5 hours at 25.5°C. Control eyes for the DOPA reaction were incubated without DOPA or in a DOPA medium containing 0.01% PTU as an inhibitor. Since it has been suggested (19) that peroxidase is involved in the initial steps of melanin synthesis in some systems, some eyes were also incubated in the diaminobenzidine medium of Graham and Karnovsky, as described by Okun et al. (19). To test this implication, some eyes were treated with 0.1% catalase in 0.t M cacodylate buffer containing 6.8 % sucrose at 25.5°C for 30 minutes and then incubated in DOPA medium also containing catalase. After the various incubations, all tissues were washed in two changes of cold 0.1 M phosphate buffer containing 6.8% sucrose for a total of 15 minutes and postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer, also containing 6.8% sucrose. All tissues were embedded in Epon and sectioned with a Porter-Blum MT-2 ultramicrotome, stained with uranyl acetate followed by lead citrate, and examined with a Hitachi HU-11E electron microscope operated at 75 kV. To obtain three-dimensional perspective, serial sections were analyzed by tracings from electron micrographs. RESULTS
D OPA reaction in normal pigmented epithelial cells The m o r p h o l o g y of the pigmented epithelium of n o r m a l a n d P T U - t r e a t e d R.
pipiens a n d X. Laevis larvae has already been described (5, 7). The larvae used for FIG. 2. Electron micrograph illustrating the intricate system of DOPA-positive tubules (7") which link clusters of late premelanosomes in Rana pipiens, x 75 000. Fro. 3. The interconnecting DOPA-positive tubules (T) are present but less extensive in Xenopus laevis. Note the scalloped appearance of the boundary of the late premelanosomes, x 75 000.
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the present experiments were younger than those studied previously. The only differences noted were the absence of myeloid bodies and the more frequent appearance of Golgi complexes and early premelanosomes. When the tissue has been incubated in the DOPA medium, electron-dense reaction product is observed in the cisternae of the distal or active face Of the Golgi apparatus and occasionally in coated Golgi vesicles (Fig. la). Although the pigmented epithelial cells are polarized, so that the melanosomes are primarily in the apical cytoplasm, the active face of the Golgi apparatus may lie el[her toward or away from the apical surface of the cell (Fig. 1 a). No reaction product is observed in early premelanosomes (Fig. 1 b); but it is difficult to ascertain whether electron dense material in intermediate and late premelanosomes is reaction product or naturally deposited melanin. The peripheries of some premelanosomes in eyes incubated in DOPA medium often have a scalloped appearance (Figs. 1 a and 3) which is absent in the controls, suggesting that DOPAreaction product is deposited in this region. According to Fitzpatrick et al. (8), melanosomes are complete organelles containing little or no tyrosinase activity. Therefore, the scalloped structures, although relatively homogeneous in electron density, are presumably late premelanosomes. The melanosomes of more mature melanocytes are not scalloped after incubation in DOPA medium. DOPA-reaction product is also present in an anastomosing system of tubules which interconnect premelanosomes and developing complex melanosomes (Figs. 2 and 3). In addition, reaction product was observed in continuities directly linking the Golgi apparatus with both premelanosomes and developing complex melanosomes (Figs. 4 and 5). Occasionally, late premelanosomes are encompassed in an intricate DOPApositive network which appears in the form of loops in individual micrographs. However, serial sections show that these looped structures are continuous through 5-8 sections and must, therefore, extend at least 300 nm (Fig. 6). These broad structures are therefore considered to be intricately folded cisternal structures which merge with the premelanosome membrane in several places and are continuous with the DOPA-positive tubules linking other premelanosomes.
D OPA reaction in pigmented epithelial cells of larvae reared in phenylthiourea (PTU) The DOPA reaction, as well as melanin synthesis can be inhibited by PTU, presumably because P T U binds with copper ions which are essential for tyrosinase FIG. 4. Four serial sections (total thickness -0.12 p) illustrating the relationship of developing complex melanosomes (C) and elongated late premelanosomes (E) to cisternae of the Golgi complex (6) in Rana pipiens. The arrow (Fig. 4c) suggests a direct continuity, x 36 000.
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activity (12). However, to ascertain the localization of tyrosinase in the pigmented epithelium of PTU-treated larvae, an attempt was made to remove the P T U from the fixed tissues and hopefully reactivate the tyrosinase. This was done by washing the tissue overnight in DOPA medium at 4°C and reincubating in fresh D O P A medium at 25.5°C. Although the extent of PTU removal is uncertain, the washed pigmented epithelial cells of PTU-treated larvae nevertheless contained reaction product in the Golgi apparatus and associated smooth membrane systems (Fig. 7), whereas unwashed cells did not contain any reaction product. In the washed cells, little reaction product was found in premelanosomes which would normally develop to form elongate melanosomes, nor was reaction product associated with oocyte melanosomes which would normally become complex melanosomes. Controls
No DOPA-reaction product was found in normal eyes incubated in medium-con taining P T U or in substrate-free medium. In addition, non-pigment-producing cells of eyes incubated in DOPA medium contained no reaction product. No peroxidase reaction product was observed in tissues incubated in diaminobenzidine; and catalase in the DOPA medium did not affect the accumulation of DOPA-reaction product in the cells. These results suggest that peroxidase plays no role in the formation[of the DOPA reaction product observed in these amphibian pigmented epithelial cells. DISCUSSION Our results confirm that tyrosinase is localized in elements of the Golgi apparatus and that early premelanosomes contain little or no demonstrable tyrosinase. Both premelanosomes and developing complex melanosomes are linked to the Golgi apparatus by DOPA-positive, smooth-surfaced channels. In addition, both are interconnected by an anastomosing network of DOPA-positive tubules. Some premelanosomes are encompassed by intricately folded, DOPA-positive cisternal elements, which merge with the premelanosome membrane and interconnect with tubules joining other developing melanosomes. Our interpretation of some possible forms and associations of the network is illustrated in Fig. 8. Since this network appears to be greatly reduced in older cells, it may be a transient structure. However, it is difficult to ascertain whether the system itself is transient or merely ceases to be DOPA positive. FIG. 5. Adjacent serial sections showing continuity (arrows) of late premelanosome and cisternae of Golgi complex (G) in Rana pipiens, x 44 000. Ft6. 6. Serial sections (total thickness = 0.3 #) demonstrate in Rana pipiens the intricately folded system of DOPA-positive cisternae which encompass some late premelanosomes. Note connections with the late premelanosomes (arrows). x 36 000.
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The presence of DOPA-reaction product in a particular location in a fixed cell does not confirm that tyrosinase is active at that site in the living cell. For example, it may be that intracellular tyrosinase inhibitors are unbound, washed out, or inactivated during fixation, thus activating the tyrosinase. On the basis of his ultrastructural and biochemical studies, Seiji (23) suggested that although tyrosinase can be demonstrated in membrane as well as premelanosome fractions isolated from melanomas, actual melanin synthesis occurs only in premelanosomes which have received their quantum of tyrosinase before separation from the Golgi appartaus. Membrane digestion studies by Seiji (23) suggest that tyrosinase may be so tightly bound to isolated smooth-surfaced membranes that it is actually an integral part of the membrane. Yet, those smooth-surfaced membranes did not contain significant amounts of radioactivity after animals were injected with 14C-DOPA; only the fractions which contained premelanosomes were significantly radioactive. It seems possible, however, that melanin precursors not bound to the membranes may have been washed out during the membrane isolation procedure. This would account for the interpretation that melanin synthesis occurs only in premelanosomes. Complex melanosomes in the pigmented epithelium of larval amphibians seem to provide an additional complication for Seiji's hypothesis that premelanosomes form directly from smooth-surfaced, tyrosinase-containing vesicles derived from the Golgi apparatus. The complex melanosomes are formed by the deposition of new melanin around oocyte melanosomes which in this environment may be regarded as premelanosomes. Thus, in this case these premelanosomes preexist in the cell and are clearly not formed from tyrosinase-containing Golgi vesicles. Our evidence shows that developing complex melanosomes, as well as premelanosomes, are connected directly to the Golgi complex by smooth-surfaced, DOPA-positive channels-channels through which enzyme is transported. An experiment was designed to provide further insight into the question of where tyrosinase is active in the living cell. This experiment utilized the eyes of larvae reared in P T U to inhibit melanin synthesis. Premelanosomes are formed in the pigmented epithelium of such PTU-treated larvae, but no pigment is deposited on them (5). In PTU-treated larvae these premelanosomes appear as early premelanosomes, but under normal circumstances they would have become melanized. We proposed two possibilities for the localization of DOPA-reaction product in cells from which all or some of the PTU was removed by washing prior to incubation in DOPA medium. The two possibilities are: (a) If the tyrosinase passes through the channel linking the
FIG. 7. An electron micrograph of pigmented epithelium from Rana pipiens larvae reared in PTU. PTU was washed out of tissue prior to incubation in DOPA medium. The reaction product is localized in tubules and cisternae but not in premelanosomes (P). x 10 900.
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Golgi apparatus and premelanosomes to become active only at this site in the living cell, then it should accumulate there. In this case, DOPA reaction product would be dense in the premelanosomes after removal of PTU. (b) If tyrosinase is not active primarily in the premelanosomes then enzyme activity should be localized in the linking channels of the PTU-treated cells. The latter proved to be the case. Most of the reaction product was localized in the linking membrane system rather than in the premelanosomes. This experiment suggests that tyrosinase activity and, therefore, melanin synthesis at least begin in the DOPA-positive channels which link the Golgi apparatus with premelanosomes. Some evidence indicates that at high concentrations PTU slightly inhibits protein synthesis (11). If the net synthesis of tyrosinase is affected by the concentration of PTU used in rearing the larvae, it could present some serious problems in the interpretation of our experiment. It has been shown, however, that concentrations of PTU greater than that used in our experiments do not inhibit the synthesis of tyrosinase in ascidian embryos (28). In addition, it is unlikely that tyrosinase synthesis and intracellular transport are seriously affected by our procedure, since the larvae as a whole develop normally (except for pigmentation) in PTU. Autoradiographic studies tracing the incorporation of 3H-DOPA into melanoma melanocytes led Zelickson et al. (10, 29) to suggest that melanin or its immediate insoluble precursors are present in the smooth endoplasmic reticulum. They propose that tyrosinase is active within the membrane system as well as in the premelanosomes. Our results support this hypothesis by suggesting that in the living cell tyrosinase is active in the DOPA-positive channels which link the Golgi complex and premelanosomes. If this is true, melanogenesis involves the dynamic process of enzyme condensation in the Golgi apparatus, biosynthesis of melanin beginning in the DOPA-positive channels and deposition occurring in premelanosomes. The relative proportion of melanin synthesis in the various cellular compartments, however, is still open to question. It is possible that as the premelanosomes mature, tyrosinase is transferred to them; thus, progressively more melanin synthesis may occur in the intermediate or late premelanosomes. Apparently some melanogenesis occurs in these premelanosomes, as evidenced by the scalloped appearance of their peripheries after incubation in DOPA medium. The transfer of material to the premelanosome via channels from the Golgi complex is a variance from the usual mode of operation proposed for the Golgi apparatus. In most systems it apparently functions by condensing and transferring material received from rough endoplasmic reticulum (1). The condensed material is sequestered in the cytoplasm by the pinching off or blebbing of vesicles from the Golgi cisternae. Some of these vesicles may then fuse to form larger vesicles or granules. However, Reddy and Svoboda (21) have demonstrated "numerous continuities (1) between
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FIG. 8. Illustration of some of the possible intricate configurations and associations of the DOPA-
positive membrane system. These smooth-surfaced membranes may be in the form of tubules (T) or cisternae (C). Some portions have been "cut away" (arrows) to illustrate how some of the "loops" and connections, as seen in electron micrographs, may occur in three-dimensional perspective. P1 is a late premelanosome with its bounding membrane intact, while P2 shows a naked melanosome with the associated networks of DOPA-positive cisternae cut away. A complex melanosome (CM) is shown conneted to P2 and the active face of the Golgi apparatus (G) by DOPA-positive tubules.
the limiting membranes of microbodies and rough and smooth endoplasmic reticulum and (2) between two adjacent microbodies or more" in liver cells of acatalasemic rats treated with chlorophenoxyisobutyrate (CPIB). Administration of CPIB induces a rapid proliferation of microbodies in the liver. Reddy and Svoboda suggest that the microbody connections may allow an interchange of material relative to rapidly altering protein pools. In like manner, the interconnecting network of D O P A positive tubules and cisternae linking developing premelanosomes may be established to facilitate the rapid maturation of a large number of premelanosomes during differentiation in the pigmented epithelium. In this connection, our results as well as those of Maul (13) indicate that connections between smooth endoplasmic reticulum and the premelanosomes were seen less frequently as melanization of premelano-
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somes neared completion. It seems probable, therefore, that such interconnecting membrane systems are broken down after their role in melanogenesis is completed. The authors wish to thank Gay Ann Eppig for the preparation of Fig. 8.
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BEAMS,H. W. and KESSEL, R. G., Int. Rev. CytoL 23, 209 (1968). BREATHNACH,A. S. and POYNTZ, S. V., J. Anat. 100, 549 (1966). BREATHNACH,A. S. and WYLIE, L. M., J. Ultrastruct. Res. 16, 584 (1966). DROCHMANS,P., Advances in Biology of the Skin, The Pigmentary System, Vol. VII, p. 169. Pergamon Press, London, 1967. 5. EPVlG, J. J., JR., Z. Zellforsch. Mikrosk. Anat. 103, 238 (1970). 6. - J. Embryol. Exp. Morphol. 24, 447 (1970). 7. - J. Exp. Zool. 175, 467 (1970). 8. FITZPATRICK,T. B., QUEVEDO, W. C., LEVENE, A. L., McGOVERN, V. J., MISHIMA, Y. and OETTLE,A. G., Science, 152, 88 (1966). 9. GUTTES,E. and BRANDT, S. M., J. Histochem. Cytochem. 9, 457 (1961). 10. HIRSCH, H. H., ZELICKSON,A. S. and HARTMANN,J. F., Z. Zellforsch. Mikrosk. Anat. 65, 409 (1965). 11. KITANO,Y. and Htr, F., Exp. Cell. Res. 64, 83 (1971). 12. LERNER,A. B., CALKINS,E. and SUMMERSON,W. H., J. Biol. Chem. 187, 793 (1950). 13. MAUL, G. G., J. Ultrastruct. Rex. 26, 163 (1969). 14. MAUL, G. G. and ROMSDAHL,M. M., Cancer Res. 30, 2782 (1970). 15. MAt~L, G. G. and BRt~MBAU~H,J. A., J. Cell Biol. 48, 41 (1971). 16. MOYER,F. H., Amer. ZooL 6, 43 (1966). 17. NIEUWKOOP, P. D. and FABER, J., Normal Table of Xenopus laevis (Daudin). North Holland Publ., Amsterdam, 1956. 18. NOVtKOEE,A., ALBALA,A. and BIEMVlCA,L., J. Histochem. Cytochem. 16, 299 (1968). 19. OKUN, M. R., EDELSTEIN,L. M., OR, N., HAMADA,G., DONNELLAN,B. and LEVER,W. F. Histochemie 23, 295 (1970). 20. RAPPAPORT,H., NAKAI, T. and SWIFT, H., J. Cell. Biol. 16, 171 (1963). 21. REDDY, J. and SVOBODA,D., Lab. Invest. 24, 74 (1971). 22. RODRIGUEZ,H. A. and McGAVRAN, M. H., Amer. J. Clin. Pathol. 52, 219 (1969). 23. SEre, M., Advances in Biology of the Skin, The Pigmentary System, Vol. VIII, p. 189. Pergamon Press, London, 1967. 24. SHUMWAY,W., Anat. Rec. 78, 139 (1940). 25. STANKA,P., Mikroskopie 26, 169 (1970). 26. - Z. Zellforsch. Mikrosk. Anat. 112, 120 (1971). 27. WELLINGS,S. R. and SEIGEL,B. V., J. Ultrastruct. Res. 3, 147 (1963). 28. WHITTAKER,J. R., Develop. Biol. 14, 1 (1966). 29. ZELICKSON,A. S., HIRSCH, H. M. and HARTMANN,J. F., J. lnvest. DermatoI. 45, 458 (1965).