Internal Structure of Avian Melanin Granules : An Electron Microscope Study

Melanin granules represent an interesting type of separate and characteristic intracellular particle formed by specific cells. While much attention has been devoted to the chemistry of the formation of the pigment, relatively little has been directed to the particles themselves, probably because they lie on the border of resolution of the optical microscope. Capacity to secrete these granules may sometimes be lost, but is regained by a mechanism which has some analogies to an infective process. This has been described for grafts of mammalian skin by Billingham and Medawar (1948), and a somewhat similar process may have occurred in the case of human melanoma described by Botha and Lennox (1954). In their case the melanoma metastases, as often happens, had lost the ability to form pigment, but this had been regained when they came into contact with the skin. Unlike the plant enzyme, mammalian tyrosinase is associated with large ultramicroscopic particles, which may even be the actual melanin granules themselves.

It is known that melanin particles may differ in shape in various species, but their exact differentiation from adventitious dirt is not easy. This is shown, for example, in the account of the granules in various breeds of fowl by Bohren, Conrad, and Warren (1943). Mason and his colleagues studied mammalian granules (from cattle, mice, and man) by the electron microscope, and concluded that they were definite ‘formed elements’ but that they had no internal structure ; no great differences in size or shape were found among the various granules.

An investigation of the melanin granules of the fowl by the electron microscope showed that, unlike the mammalian particles, some of them had a complex internal structure, and the various types differed very much in both form and size. Furthermore, it has been found possible to examine the particles directly in the feather by the electron microscope. This opens the way to a study of the genetic and racial variations, as well as the endocrine-induced changes, by morphological means. As it is not intended to pursue this type of study, a description of the methods and results so far obtained may be of value to other workers.

Either skin with growing feathers, clipped free of fully formed feather, or the roots of growing feathers plucked from the skin were used. The material was macerated with saline and the particles concentrated and washed by centrifuging ; the coloured layer of granules was easily followed through the processing. After washing and purifying by a few cycles of centrifuging, a suitable dilution was mounted on electron microscope grids covered with collodion in the usual way. Examination was by a Philips 25A electron microscope ; any shadowing was done with gold-palladium alloy.

The particles from the black breast feathers of the male Brown Leghorn were found to consist of dense rounded rectangles or lenticules about 1 p. long, with a smooth and sharply defined edge and surface. They have a well-defined exterior wall, about one-quarter of the breadth of an average granule ; this is quite structureless. The central space may contain, rarely, a few ‘vacuoles’, but is more usually filled with a single row of reticulated material (fig. 1 A), or sometimes two or three rows (B). This latter condition is usually seen in the more lenticular granules ; it gives a central, or more infrequently subterminal, bulge (c). Occasionally the central space seems almost empty, containing only a few thin transverse strands. Shadowed preparations indicate that the surface is smooth, and that the granules are also lenticular in cross-section, with the greatest thickness about one-half of the breadth. Sometimes abnormally long forms are found. These appear to be almost double granules, arranged end to end (D), perhaps with only a small portion of the structureless wall in common. These forms suggest either multiplication by self-reduplication or in sequence from a generation point. The extreme diversity of forms was quite striking.

That this internal structure is not an artifact of preparation is indicated by the fact that exactly similar pictures were obtained when the extracted material was immediately fixed by buffered formalin or osmium tetroxide before separation, and also by the results of direct examination of the feathers, as described below.

It was found, rather surprisingly, that the particles could easily be examined directly by the electron microscope in fully formed feathers. A single barb or barbule is washed in acetone, dried, and mounted across a roo-mesh copper grid. When inserted into the microscope vacuum, the feather ‘explodes’ and shears along the keratin layers. Further shearing occurs in the heat of the electron beam, leaving the granules mounted for examination on a very thin layer of keratin that is relatively transparent to the electron beam and almost structureless, at the magnifications used. Naturally, under these conditions the resolution is not very good, as the static charges on the feather cause some distortion of the electron beam, and also the preparation is not usually perfectly stationary. Details of the interior of the black granules can still be made out, however, and the actual preparations, examined with continual adjustments of focusing and beam-centring, give a much better idea of the structure than the photograph (fig. i, E) indicates, since the exposure needed for photography is too long for the unstable conditions of the preparation.

Observations by this method of the black male feathers confirmed that the smooth surface, internal structure, shape, and thickness were as deduced from the shadowed preparations of free particles. An impression was gained that the particles, arranged in lengthwise rows, contributed quite notably to the bulk and strength of the feathers.

White feathers, or patches of white found in feathers, often occurring in fowls, were found to be quite devoid of granules. Occasional inclusions of dirty material were quite easily distinguished from granules.

Rhode Island Red feathers examined by the same direct method contained granules which were obviously quite different in structure. These were discoidal, and with no internal structure, very uniform in size, and closely resembled the mammalian granules described by Mason and his colleagues (1947). Not one looking like the black male Leghorn type was seen. The down and female breast feathers of Brown Leghorns contained both types of granules, but the various areas examined were usually clearly of one type or the other, and not mixed.

When examined as a preparation of free particles, Rhode Island Red granules were seen to be roughly rounded or commashaped particles, about one-third of the length of the black Brown Leghorn ones, having a rather rough crenated wall and only a hint of an internal structure (fig. 1, F, G). Compared with the former they strongly suggested a badly made and imperfect product, which is not surprising as they are the end-result of a mutation from the normal. As in the case of the black granules, they were arranged in the feather in long strands, with the edges almost touching.

Needless to say, this method of ‘exploding’ the feathers in the microscope resulted in a certain amount of debris being left in the instrument. On cleaning, numerous fragments of feather were found in the object cavity. Fortunately, the Philips microscope is very tolerant of dirt in this position, though its performance was doubtless somewhat impaired by the presence of this material. No feather particles were found elsewhere. However, as the objective of the Philips microscope can only be cleaned after a complete dismantling of the whole microscope column, it seems preferable that another type of instrument should be used for such work.

The secretion of melanin pigments in the fowl in the form of granules is clearly an elaborate process terminating in the formation of a particle of complicated structure,’ and not merely the production of an insoluble lump of protein-melanin complex. The change from black to red—which is under hormone control—is not merely one of chemical structure, but also of the gross structure of the granule itself. It would therefore appear that the avian material offers especially favourable material for such studies; for the basic type of granule is a well-defined and easily recognizable structure, differing from any other reported type of cell organelle. The problem of the extra-epidermal origin and the development of granule-secreting properties in the cells of the neural crest of the embryo (Dorris, 1945 ; Rawles, 1945) can thus be favourably studied in well-known and easily available material. The fact that avian granules have a recognizable and characteristic structure suggests that further comparative studies should be interesting. Melaninization is found in most of the Metazoa, and a study of this by micro-morphological methods may be worth while. Methods analogous to those described for the use of very small pieces of whole feather could doubtless be used on museum specimens without causing any particular damage.

All expenses in connexion with this work were borne by the British Empire Cancer Campaign.