It is now agreed by most observers that melanin formation in all vertebrates takes place in specialized dendritic cells called melanocytes. The amount of melanin in the skin and hair is subject to considerable variation and is dependent on the numbers of melanocytes present and on the activity of these cells. Ever since Bloch’s discovery of the Dopa reaction (1917) numerous investigations have been carried out in an attempt to explain the mechanisms involved in regulating the production of melanin. We are interested in studying the effect of the sex hormones on melanocytes and melanin production using carefully controlled histochemical experiments, and for this purpose we have chosen the guinea-pig as a suitable
laboratory animal. Before investigating the effect of the male sex hormone on melanogenesis in the male guinea-pig, it was considered fundamental to the problem that a thorough knowledge of the numbers and morphology of the melanocytes and the amount of melanin present in the different skin regions in the normal animal at different stages of sexual development should first be obtained. During the course of this investigation we have also closely studied the precise relationship of the melanocytes and melanin granules to the other cells of the epidermis. To simplify the investigation we have confined our study to the melanocytes and melanin found
in the surface epidermis, and have ignored completely the melanocytes and melanin of the hair follicles. The results of this preliminary work form the substance of this paper.
MATERIALS AND METHODS
Pure black and pure red male guinea-pigs were used and these were divided into
three groups: (1) Twenty-four mature animals all weighing between 450 and 912 g.
to study the melanocytes in the normal adult guinea-pig. (2) Seven immature
animals all weighing under 265 g. and known to be less than 25 days old to study the
melanocytes before the onset of sexual activity. (3) Seven animals weighing between
350 and 890 g. to study the melanocytes during the period of rapid sexual development (Deanesly & Rowlands, 1936).
Skin samples measuring about 0 5 cm.2 were removed from all the animals from
the ear, anterior abdominal wall and sole of foot; in addition the right areola was
excised. Most of the skin samples were left intact after excess subcutaneous fat and
the deeper part of the dermis had been excised under a dissecting microscope. A few
of the samples were treated with trypsin to disengage the epidermis using the ‘skinsplitting’ technique of Billingham & Medawar (1951), as modified by Szabo (1955).
All the samples were then processed under identical conditions. The skin was fixed
in 5 % formol saline for 16 hr. and after washing it in normal saline for i hr. it was
incubated at 370 C. in a 1 in 1000 solution of L-dihydroxyphenylalanine at a pH
P. G. Bi8chitz and R. S. Snell
of 74. After 2 hr. the substrate was renewed and incubation continued for a further 1I hr. Fixation for a further period of 16 hr. in 10 % formol saline was then carried out, followed by dehydration and clearing. The greater part of each skin sheet was mounted in Canada Balsam, the remainder was embedded in paraffin and vertical sections 6 p thick were cut. In order to identify the various layers of the epidermis a number of the vertical sections were counterstained with haematoxylin and eosin. All the skin sheets were examined with the dermal surface uppermost except those of the anterior abdominal wall where, owing to the large number of hair roots present in the dermis, it was found more satisfactory to examine the sheets with the epidermis uppermost. The appearances of the melanocytes in the skin samples removed from the different groups of animals were studied by assessing the number and size of the melanocytes and the amount, colour and position of the melanin within the cells. The length, width and complexity of the dendritic processes were also noted and an attempt was made to assess the amount and colour of the free melaidin present, i.e. that melanin which is situated outside the melanocytes. The skin sheets of the sole of the foot were examined only in the red animals, since the very high concentration of melanin present in the skin of the black animals in this region prevented their accurate assessment. The melanocytes were counted by projecting the image of a skin area measuring 004 mm.2 on to a ground-glass screen marked out in the form of a grid. Ten randomly
chosen areas were counted at a magnification of 375. Black skin was less easy to
count than red, because it possessed more and darker free melanin granules which
tended to obscure the melanocytes. In the sole of the foot melanocyte counts were
found to be impracticable owing to the melanocytes being superimposed one upon
the other on the sides of the very steep dermal papillae.
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