Pigment cell development in rhesus monkey eyes: an electron microscopic and histochemical study

DEVELOPMENTAL

Pigment

BIOLOGY 32, 69-81 (1973)

Cell Development Microscopic

in Rhesus Monkey and Histochemical

HIDEHIKO Department

of Cutaneous Biology,

Eyes: an Electron Study’

ENDO AND FUNAN Hu

Oregon Regional Accepted

Primate

November

Research

Center,

Beauerton,

Oregon 97005

9, 1972

The ontogeny of pigment cells in the eyes of rhesus monkeys was studied by electron microscopy and histochemistry. In 60. to do-day-old fetuses, the pigment epithelium of the iris and retina has already differentiated whereas stromal melanocytes of the uveal tract differentiate much later. The morphological and histochemical difference between melanocytes of the iris stroma and the choroid suggests that during embryonic development melanocytes migrate from the iris toward the ciliary body and choroid. Similarly, melanosomes of pigmented epithelial cells may have their origin in the epithelium of the anterior layer of the iris, which is metabolically more active than both the posterior layer and the pigment epithelium of the ciliary body and retina.

of rhesus monkey eyes during development and to confirm the observation made earlier by Hu and Montagna (1971). Fitzpatrick and Quevedo (1971) recently described melanosome development in four stages (I, II, III, and IV)Z according to the degree of melanization. We will follow their classification because it enables us to define the functional and structural changes of melanosomes during ontogenesis.

INTRODUCTION

The eyes of rhesus monkeys possess two lines of pigmented cells: those of the pigment epithelium and those of the uveal tract. The two lines differ in origin, morphology, and time of development. Hu and Montagna (1971) have recently shown by light microscopy and histochemistry that the pigmented epithelial cells differentiate early and are functional in 65 day-old fetuses, whereas stromal pigmented cells are not recognizable until late gestation. They have also shown that the distribution and enzyme activity of the stromal pigment cells change during development. The total absence of strongly DOPA-reactive dendritic cells in the choroid of fetuses of all ages suggests that propigment cells originate in the iris and migrate to the choroid late in ontogeny. The purpose of this ultrastructural study is to investigate morphological and histochemicai changes in the pigment cells

MATERIALS

Immediately after anesthetization, the eyes of prenatal (60-, 80-, 106-, 141-, 145-, and 154-day fetuses) and postnatal (16 months old and adolescent) rhesus monkeys were collected and dissected into the 2 Melanosome stage I is a spherical, membranedelineated vesicle that can be assumed to be a melanosome if it (a) is shown by electron microscopy and histochemistry to contain tyrosinase or (b) czntains filaments that have distinct periodicity (100 A). Melanosome stage II is an oval organelle which contains numerous membranous filaments, with or without cross-linking and having distinct periodicity (100 A). Melanosome stage III is an oval organelle in which the internal structure, characteristic of stage II, has become partly obscured by electron-dense melanin. Melanosome stage IV is an organelle in which melanin deposition obliterates the internal structure characteristic of stages II and III.

I Publication No. 641 of the Oregon Regional Primate Research Center supported in part by Public Health Service, National Institutes of Health Grants RR 00163 of the Animal Resources Branch, Division of Research Resources, AM 08445 of the National Institute of Arthritis and Metabolic Diseases, and CA 08499 of the National Cancer Institute.

69 Copyright All rights

0 1973 by Academic Press. Inc. of reproduction in any form reserved.

AND METHODS

70

GEVELOPMENTALBIOLOGY

iris, ciliary body, and choroid (Hu and Montagna, 1971). All tissues were fixed in cold 4% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 for 1 hr. While in fixative, the tissue was cut into about 1 mm3 pieces, which were incubated in 0.1% L - 3,4 - dihydroxyphenylalanine (DOPA, Sigma Chemical Co.) in 0.1 M phosphate buffer at pH 7.4 for 5 hr at 37°C and postfixed for 90 min in cold 1% osmium tetroxide in Verona1 acetate buffer at pH 7.4. Control sections were incubated in the

VOLUME 32, 1973

buffer without DOPA and processed in an identical manner. The pieces were then washed, dehydrated in graded alcohol, and embedded in Epon. Ultrathin sections (100 nm) were cut with a Porter-Blum MT2 microtome, stained with uranyl acetate and lead citrate (Reynolds, 1963), and examined with a Philips 200 electron microscope. OBSERVATIONS

The observations Tables 1 and 2.

are summarized

in

TABLE 1 PIGMENT EPITHELIUMS

Melanosome Prenatal 60 Days

80 Days

Iris, anterior

Retina

Age of monkey

I II III IV I II

+ ++ +++ +++ + ++

DOPA

+

+

III +++ IV +++ 106 Days

141Days

I II III IV I II

~ ~ ++ +++ +

+

+

III ++ IV +++t 145 Days

154 Days

Postnatal 16 Months

I II III IV I II

+ + +++

+++t ~ ~

III

++

IV

+++t

I II III

+

IV +++ Adolescent

I II III

IV

+ t+

+

-

Melanosome

I II III IV I II III IV I II III IV I II III IV I II III IV I II III IV I II III IV I II III IV

+ + +++ + +

layer

Iris, posterior

DOPA

+ ++ +++ + +++

layer DOPA

+

+

t;t+ +++ ++ +++ ++ ++++ ++ ++++ + +++

Melanosome

I II III IV

+ ++

I II III IV I II III IV I II III IV

~ + +++ + +++ ~ +++

I II III IV I II III IV

~ ~ ++++ ~ ++++

++

+

+

i

+

“Roman numerals I-IV indicate stages I-IV, respectively, Melanocyte, melanosome: -, absent; +, present, a few only; ++, moderate numbers; +++, many; ++++, a great many. DOPA reaction: -, negative; +, questionable; +, weakly positive; + +, moderately positive; + + +, strongly positive.

ENDO AND Hu

Pigment

Cell Deuelopment

71

in Eyes

TABLE 2 STROMAL MELANOCYTEV Age of monkey

Choroid Melanocyte

Prenatal 60 Days

80 Days

-

106 Days

+

141 Days

+

145 Days

+

154 Days

++

Melanosome

I II III IV I II III IV I

-

II

+

III IV I II III IV I II III IV I

+ + + + -

Iris DOPA

Melanocvte

-

+

-

+

+

++

+

+++

+

+++

-

+++

-

+++

II + III + + IV +++ Postnatal 16 Months

Adolescent

a See footnote

Development

+++

+++

I II III IV I II III IV

++++ ++++

+++

Melanosome

I II III IV I II III IV I 11 111 IV 1 II III IV I II 111 IV I II III IV

+ ++ + + ++ + +++ +++ + +++ +++ +++ +++ +

I II III IV I II III IV

+ + ++ + +++ +

DOPA

+

+++

+++

++

++

+

+

a of Table 1.

of Pigment

Epithelium

Retinal pigment epithelium. The retinal pigment epithelium consists of a single layer of cuboidal cells, already well differentiated in 60- to BO-day fetuses. The layer is grossly pigmented, with cells containing many stage II, III, and IV melanosomes in various shapes-round, ovoid, or spindle-and sizes, about 0.8-2 pm in length and 0.2-0.9 pm in diameter (Fig. la). Stage I melanosomes are seldom found. All melanosomes have a limiting membrane and stage II melanosomes

have a characteristic internal structure of arrays of membranes with a regular periodicity (Fig. lb). Melanosomes in the pigment epithelial cells of the log-day fetus resemble those of 60- and go-day fetuses. The number of stage IV melanosomes increases in 141-, 145-, and 154-day-old fetuses and after birth tends to decrease (Table 1). In all the fetuses examined, some DOPA reaction product is present in Golgi cisternae, smooth vesicles, and endoplasmic reticulum (ER) close to the Golgi complexes (Fig. 4). Whether DOPA re-

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DEVELOPMENTALBIOLOGY

VOLUME 32, 1973

FIG. 1. Retinal pigment epithelium of an 80.day fetus. (a) Note many melanosomes of stages II, III, and IV (m2 to m4) in various shapes and sizes, Golgi apparatus (g), and terminal bar (arrow) between adjacent cells. x 14,300. (b) Note a stage II melanosome and a stage IV melanosome. The former shows regular periodicity. x 96,000.

ENDO AND Hu

Pigment

action product is present in the melanosomes is not clear. No DOPA reaction was detected in postnatal eyes. Melanosomes begin to decrease in number after birth. Lysosomal dense bodies were first observed in the pigment epithelial cells of a 16-month-old monkey. These spherical or ovoid bodies, about 0.5-0.8 pm in diameter, have a finely granular matrix limited by a single membrane and often contain irregularly dense or lamellated materials which increase in number as the animal approaches adolescence (Figs. 2b, 2~). Other cytoplasmic organelles also vary somewhat at different fetal stages. Free ribosomes, which occur singly or as polysomes, are numerous in early fetuses, but less so in later stages. Golgi complexes are well developed at all fetal stages, but not after birth. The amount of rough endoplasmic reticulum (RER) decreases after birth, whereas smooth endoplasmic reticulum (SER) tends to increase and become very prominent in postnatal animals (Fig. 2a). The cytoplasmic process in the apical surface of the retinal pigment epithelium becomes prominent in late fetal stages and develops extensively after birth. A basal lamina separates the retinal pigment epithelium from the vascular choroid. Terminal bars are present between adjacent plasma membranes (Fig. la). Iridiul pigment epithelium. Iridial pigment epithelium has an anterior and a posterior layer of pigmented cells. Basal lamina is present on the free surface of the posterior layer and the basal surface of the anterior layer. The plasma membranes of adjacent cells and those between cells of the anterior and posterior layers have junctional complexes. In the fetus, the pigment epithelial cells in both layers are cuboidal or low columnar. In the neonate, however, the cells in the posterior layer become larger and columnar whereas those in the anterior layer become smaller and cuboidaf.

Cell Development

in Eyes

73

During both fetal and postnatal life, melanosomes in the cells of the anterior layer vary in degree of melanization and in size and shape (Figs. 3b, 3c, 3d). By contrast, those in the posterior layer are more uniform and are generally larger and more fully developed (Fig. 3a). The long spindle-shaped melanosomes characteristic of the retinal pigment epithelium are rarely seen in the iris. In both fetal and postnatal eyes, melanosomes range from 0.8 to 1.4 pm long and 0.5 to 0.8 pm wide. Stage III melanosomes, common in the anterior pigment layer of 60- or 80-day-old fetuses, are replaced by increasing numbers of stage IV in late fetal and postnatal stages, whereas in the posterior pigment layer stage IV melanosomes predominate during all stages of development (Table 1). The number of melanosomes in the iridial epithelium of fetal and postnatal eyes also varies, especially in the posterior layer; the few scattered melanosomes in the 60- or go-day-old fetus increase manyfold in the l&month-old juvenile. DOPA reaction product is found in the Golgi areas and in some melanosomes of the anterior epithelial cells throughout all fetal stages and even in trace amounts in the 16-month-old animal. In a 60-day fetus, when the smooth vesicles and ER near the Golgi areas contain reaction product, they resemble the so-called stages I and II melanosomes, measuring about 80 to 100 nm in diameter but varying in length (Fig. 5). However, in the posterior epithelium, only weak reactivity was found in an SO-day fetus. Except for some difference in the melanosomes, other cytoplasmic organelles are similar in the anterior and posterior pigment layers. In the early stages, cytoplasm is rich in free ribosomes. The Golgi complexes, well developed in all fetal stages, tend to be less prominent in postnatal eyes. Lysosomal dense bodies are seldom seen even in adolescent eyes. Ciliary pigment epitheliwm. The one-

FIG. 2. Retinal pigment epithelium of an adolescent rhesus. (a) Many lysosomal dense bodies but only a few melanosomes are present. Note the abundance of smooth-surfaced endoplasmic reticulum. x 11,400. (b and c) Some dense bodies (arrows) have electron-dense materials in a finely granular matrix. Compare the dense body with stage IV melanosome (m4). x 39,600. FIG. 3. lridial pigment epithelium of a 16.month-uld rhesus. (a) Arrows show the border line between anterior (bottom) and posterior layer (top). Melanosomes in the posterior layer are mostly in stage IV and rather uniform in shape. By contrast those in the anterior layer show more variation. (b, c, and d) Morphological variation and stages of melanosomes in the anterior layer. Stage 11 (m2), stage III (m’l), stage IV (m4). x 39,600. 76

FIG. 4. Retinal pigment epithelium of an 80-day fetus, DOPA. Note DOPA reaction product in GERL, smooth vesicles, and Golgi cisternae. x 62,000. FIG. 5. Iridial pigment epithelium of a 60-day fetus, DOPA. DOPA reaction product in dilated cisternal structures (arrows) which appear to contain striated membranous structures. x 62,000.

layer pigment epithelium of the ciliary processes consists of low columnar or cuboidal epithelial cells lying between a layer of nonpigmented columnar cells adjacent to the vitreous on one side and the vascular stroma on the other. It is connected to the anterior iridial pigment epithelial layer anteriorly and to the retinal pigment epithelium posteriorly. The predominantly round or ovoid melanosomes in the ciliary pigment epithelium resemble those in the posterior epithelial cells of the iris. Development of Stromal the Uveal Tract

Melanocytes

in

Choroidal melanocytes. Melanocytes containing melanosomes first appear in a 106-day fetus and are still scarce at 145 days. These melanocytes have stage II or III melanosomes which measure about 230-340 nm in length and 60-70 nm in

diameter (Figs. 6a and 6b). At 154 days they suddenly increase in number and contain stage II, III, and IV melanosomes. The stage II melanosomes are small and rod-shaped and similar in size and shape to those in the iris stroma; the stage IV granules, however, are larger and round or ovoid, measuring about 250-340 nm in length and 120-170 nm in diameter (Fig. 7). Melanocytes continue to increase in number after birth. Their cytoplasms are packed with stage IV melanosomes that are mostly round or ovoid and smaller than those of the pigment epithelium (Fig. 8). DOPA reaction products are present in 106-day (Figs. 6a, 6b), 141-day, and 145day fetuses, but not in older ones. Choroidal melanocytes are recognized by the many filaments in their cytoplasm (Fig. 7). At times, when they have only a few stage II and III melanosomes, they

FIG. 6. The choroid of a 108day fetus, DOPA. (a) Only a few melanocytes are present. They are identified by DOPA-reactive melanosomes (arrows) in their cytoplasm. x 7000. (bl DOPA-reactive melanosomes at higher magnification (arrow). x 44,000. FIG. 7. Choroidal melanocytes of a 154.day fetus, DOPA. Note stages II, III, and IV melanosomes and many filaments in the cytoplasm. x 39,000. FIG. 8. The choroid of a 16-month-old rhesus. Note many melanocytes packed with stage IV melanosomes. x 3800. 76

ENDO AND Hu

Pigment

also have well-developed Golgi complexes, many free ribosomes, and a moderate number of smooth and rough-surfaced ER. By contrast., those cells that contain mainly stage III or IV melanosomes have very few other cytoplasmic organelles, and their Golgi complexes appear less developed. Iridial melanocytes. Melanocytes are rare in 60-day fetuses. Only one, identifiable by the DOPA reaction product, was found close to the Golgi area (Fig. 11). A few melanocytes appear in go-day fetuses (Fig. 9a) and increase in number as the fetuses advance from 106 days to 154 days (Fig. 10). Melanocytes in SO-day fetuses contain mostly stage II melanosomes (Fig. 9b); those in older fetuses (106-day, 141-day, 145-day, and 154-day) have in addition many stage III granules (Fig. 10). Stage IV melanosomes are relatively scarce even in postnatal eyes (Table 2). Melanosomes in the melanocytes of the iris stroma are generally smaller and rodshaped, unlike the larger round or ovoid ones in the choroidal cells. DOPA reaction becomes prominent in 80-day fetuses, mainly in the Golgi-associated vesicles and SER (Fig. 12). Because both are electron opaque, it is sometimes difficult to determine whether the stage III or IV melanosomes have been darkened by melanization or by the DOPA treatment. The difference becomes apparent when the DOPA-treated materials are compared with those in untreated controls. In all fetal stages except the 60-day, DOPA-treated melanosomes in the iridial melanocytes always have more electron opacity than those in the untreated controls (Figs. 12 and 13). DOPA reactivity is still demonstrable in a 16-month-old animal (Fig. 14). Stromal melanocytes in the ciliary body. The characteristics of ciliary melanocytes are more or less transitional between those of the iridial and choroidal cells. DOPA reaction product is present in the Golgi-

Cell Development

in Eyes

77

associated vesicles, SER, coated vesicles, and in some melanosomes. DOPA-negative stage II melanosomes are also present along with reactive ones. DISCUSSION

Pigment Epithelium Pigment epithelium in the eyes of the rhesus monkey fetus is well differentiated at 60 days; in man it is melanized at about the fifth week of gestation which corresponds roughly to the third week in rhesus monkeys. Further development is a relative increase of stage IV melanosomes during the later fetal stages. The persistence of DOPA reactivity in the pigment epithelium up to 154 days indicates that even well-differentiated cells continue to produce new melanosomes. Retinal pigment epithelium plays a role in the metabolism of the rod outer segments of the neural retina. Rod outer segment discs are continually renewed: old discs are gradually removed from the assembly site toward the apex of the outer segment and are finally phagocytosed by the pigmented epithelial cells whose processes surround the apex of the outer segment (Young, 1970, 1971). The difference in stages between melanosomes and cytoplasmic organelles in the anterior and posterior pigment epithelium of the iris may be significant. The anterior layer shows evidence of more active metabolic activities and DOPA reactivity and has early-stage melanosomes, whereas the posterior layer has mostly fully developed melanosomes, little DOPA reactivity, and few active metabolism-related cytoplasmic organelles even in the youngest fetus examined. Therefore, either the anterior layer is relatively younger in development or it is the active site of melanosome biosynthesis. The first possibility seems less likely because the anterior layer is thought to be the continuation of the ciliary and retinal epithelium, both of which are at least

FIG. 9. The iridial stroma of an 80-day fetus. (a) A melanocyte is shown on the left. x 7000. (b) A higher magnification of the square in (a) showing stage II melanosomes. x 60,000. FIG. 10. The iridial stroma of a l&-day fetus. Note many melanocytes filled with long rod-shaped melanosomes, some melanized, some not. Round granules are cross sections of rod-shaped melanosomes. Compare with Fig. 9. Note the increase in number and in melanization of melanocytes. x 3800. 78

FIG. 11. Stromal melanocyte of the iris of a 60-day fetus. DOPA. Note DOPA reaction product. in small vesicles (arrows). x 39,009, FIG. 12. Stromal melanocyte of the iris of an 80-day fetus. Strong DOPA reaction product is seen in GERL, Golgi cisternae, vesicles and melanosomes. Some DOPA-reactive vesicles appear to bud from SER (arrow). x 44,000. FIG. 13. Stromal melanocyte of the iris of a 106-day fetus. DOPA reaction product (arrow) in an elongated stage II or III melanosome. x 39,000. FIG. 14. Stromal melanocyte of the iris of a 16-month-old rhesus. DOPA reaction product is seen in the tubular cisternae, vesicles (arrows) and melanosomes. x 39,000.

as well developed as the posterior iris epithelium in the 60-day fetus. Furthermore, in human fetuses the anterior epithelium already contains pigment by the time the iris commences to form at the 4th month. Pigmentation of the posterior epithelium begins at the pupillary margin and reaches its base at about the 6th

month (Wolff, 1968). This sequence is comparable to the development of uveal stromal pigment cells which also begins in the iris (Hu and Montagna, 1971). Thus we believe that the sites of active melanosome biosynthesis for the stromal cells are in the iris and those for the pigment epithelium in the anterior iris epithelium.

80

DEVELOPMENTALBIOLOGY

In addition to melanin, the human retina contains lipofuscin. Feeney et al. (1965) and Novikoff (1961) suggest that lipofuscin granules are derived from the lysosome-like inclusions. Similar lysosome-like inclusions are present in the pigment epithelium of the rhesus retina at 16 months of age, and more are present in adolescent and older monkeys. We believe these lysosome-like bodies represent degenerated and degraded melanin granules. They increase in number and melanosomes become fewer as the animal ages. These granules have not been seen anywhere in fetal eyes nor in the pigment epithelium of the iris in postnatal and adolescent animals. Our findings differ from those of Mund et al. (1972), who observed lipofuscin granules in the iris and choroid stroma of near-term fetuses but not in any of the epithelial layers. Feeney et al. (1965) found a number of “compound granules” with the characteristics of both melanin and lipofuscin granules; in most of them, melanin formed the medulla and lipofuscin the cortex. The existence of these intermediate forms supports the view that lipofuscin granules and lysosome-like inclusions are one and the same. They are, in fact, degenerated and degraded melanin granules. Young (1971) found lysosomal dense bodies in the retinal pigment epithelium of 8- and lo-month-old rhesus monkeys and considered them to be late stages in the digestion of the phagocytosed disc of the rod outer segment. Lysosomes are not unique to the pigment epithelial cells of the retina. They are seen in other cells and tissues where digestion and degradation take place. They are also associated with acid phosphatase activity, which Seiji and Iwashita (1965) have demonstrated in the melanosomal fractions of Harding-Passey melanoma cells. Novikoff et al. (1968), who also showed this enzyme activity in melan-

VOLUME 32, 1973

osomes of Harding-Passey melanoma, suggested that the degradation of melanosomes takes place in the autophagic vacuoles. Although lysosomal dense bodies with lamellar structure were considered to be late stages in the digestion of phagocytosed rod outer segment discs, similar structures were seen in cultured melanoma cells (Hu, unpublished data). We believe, therefore, that they are nonspecific digestion or degradation products and that they derive from either phagocytosed outer segment discs or the degradation of melanosomes or of any organelles with membranous structures. Stromal Melanocytes Throughout prenatal and postnatal development stages, the melanosomes of the iris stromal melanocytes are mostly rod-shaped and small; stage IV melanosomes are few even in adolescent eyes. Choroidal melanocytes develop later, but their melanosomes are larger than those in the iridial cells and most are fully melanized (stages III and IV predominantly). In spite of their late appearance, their maturation is always more advanced than that of the iridial cells at the same or different stages of development. Stage IV melanosomes, the predominant granules in the choroid, almost always appear round or ovoid in sections. Stage II melanosomes are rarely seen even in early fetuses when there are only a few melanocytes in the choroid; but when they are present, they are similar to, or perhaps somewhat shorter than, those in the iris. Both iridial and choroidal melanosomes appear to have the same origin and sequences of development, and apparently stromal melanocytes are first formed in the iris and then migrate posteriorly and mature in the choroid, an observation first made by Hu and Montagna (1971). This hypothesis is further supported by

ENDO AND Hu

Pigment

our histochemical data which show that tyrosinase, the enzyme for melanosome biosynthesis, is present almost exclusively in iridial melanocytes. By contrast little DOPA reaction product is seen in the choroidal cells in any stages. The existence of two morphologically distinct types of granules is intriguing. The extremely long thin granules occur only in the premelanosome stage (stage II, less often stage III) but never as fully melanized stage IV melanosomes. We believe that these long, thin granules represent early melanosomes and that as they grow their increase in size is restricted to a smaller dimension so that they become ovoid. In sections, these older, larger, and melanized granules appear round, oval, or elliptical. In older animals, the more developed granules of the iridial melanocytes are invariably thicker, and the ratio between the long and short diameters never becomes as extreme as that in young eyes. Similarly the pigment epithelial cells of the retina in the early fetus (80day) have these long, thin stage II melanosomes that disappear in older fetuses and postnatal animals and are replaced by round or oval and spindle-shaped granules. The shape and size of granules in individual animals and in different species are obviously genetically determined. The rod-shaped granules in the iridial stromal cells seen in the rhesus monkeys are not unique to this animal since similar granules have been reported in the cat (Tousimis, 1963).

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REFERENCES FEENEY, L., GRIESHABER, J. A., and HOGAN, M. J. (1965). Studies on human ocular pigment. In “Eye Structure. II Symposium” (J. W. Rohen, ed.), pp. 535-548. Schattauer, Stuttgart, Germany. FITZPATRICK, T. B., and QUEVEDO, W. C., JR. (1971). Biological processes underlying melanin pigmentation and pigmentary disorders. In “Modern Trends in Dermatology” (P. Borrie, ed.), Vol. 4, pp. 1222149. Butterworths, London, England. Hu, F., and MONTAGNA, W. (1971). The development of pigment cells in the eyes of rhesus monkeys. Amer. J. Anat. 132, 119-132. MUND, M. L., RODRICUES, M. M., and FINE, B. S. (1972). Light and electron microscopic observations on the pigmented layers of the developing human eye. Amer. J. Ophthalmol. 73, 167-182. NOVIKOFF, A. B. (1961). Lysosomes and related particles. In “The Cell” (J. Brachet and A. E. Mirsky, eds.), Vol. II, pp. 423-488. Academic Press, New York. NOVIKOFF, A. B., ALBAL.A, A., and BIEMPICA, L. (1968). Ultrastructural and cytochemical observations on B16 and Harding-Passey mouse melanomas. The origin of premelanosomes and compound melanosomes. J. Histochem. Cytochem. 16, 299-319. REYNOLDS. E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. SEIJI, M., and IWASHITA, S. (1965). Intracellular localization of tyrosinase and site of melanin formation in melanocyte. J. Invest. Dermatol. 45, 305314. TOUSIMIS, A. J. (1963). Pigment cells of the mammalian iris. Ann. N. Y. Acad. Sci. 100, 447-466. WOLFF, E. (1968). “Wolff’s Anatomy of the Eye and Orbit,” 6th ed., revised by R. J. Last, pp. 419-463. Saunders, Philadelphia, Pennsylvania. YOUNG, R. W. (1970). Autoradiographic studies on the metabolism of the retinal pigment epithelium. Invest. Ophthlmol. 9, 524-536. YOUNG, R. W. (1971). Shedding of discs from rod outer segments in the rhesus monkey. J. Ultrastrut. Res. 34, 190-203.