Browsing by Author "Kidson, Susan H"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    Open Access
    Hypomelanosis in chickens
    (1994) Marco, Heather Gaile; Kidson, Susan H
    Hypomelanosis, a severe reduction in pigmentation, is a widespread phenomenon which affects many different vertebrate species, including humans and chickens. The cause(s) of various forms of hypomelanosis is (are) not known. The aim of this study was to determine the cause of hypomelanosis in a breed of white chickens (White Plymouth Rock x Pile Game). It was hoped that this hypomelanotic breed may provide insight into the etiopathogenesis of certain human hypomelanotic disorders, such as vitiligo and albinism. To determine whether melanocytes are present in the hypomelanotic skin, two melanocyte-specific assays were carried out, in situ DOPA histochemistry and a sensitive radiometric assay for tyrosinase. The results show that active tyrosinase was present in 8, 9 and 10 day skins. However, unlike normal black skin, the level of tyrosinase did not increase with age, suggesting that the melanocytes either die or that they do not continue to synthesise tyrosinase. Ultrastructurally, these melanocytes appeared to be morphologically normal and they did not show signs of premature degeneration. Unlike black chick melanocytes, however, they contained very few premelanosomes and fully melanised melanosomes were never observed, suggesting that hypomelanosis results from the arrested development (melanisation) of melanosomes in vivo. Two different experiments were carried out to determine whether this blockage in melanogenesis is intrinsic in the melanocyte or whether it is caused by extrinsic environmental factors. The outcome of these studies were conflicting: 1) In culture, white chick neural crest cells produced pigment, suggesting that the melanocyte is not defective. However, ultrastructural examination of these cultured melanocytes showed that they contained a large proportion of partially melanised melanosomes. 2) Black chick neural crest cells migrated into white skin explants and contributed towards pigment in the developing feathers, suggesting that the white chick tissue environment is also not defective. The results hint that hypomelanosis in the white chicks is caused by the interaction of at least two genetic defects: an intrinsic mutation of the melanocyte, as well as an extrinsic mutation in the melanocyte environment that, in combination, exert an inhibitory influence on melanin synthesis. This study shows that, in situ, white chick melanocytes share some features with ty-pos albino melanocytes and may be representative of this pigmentary disorder. White Plymouth Rock x Pile Game chicks may also be useful as a model for the multi-faceted disorder, vitiligo.
  • Thumbnail Image
    Item
    Open Access
    Melanocyte proliferation in wounded skin in vitro: a histological and immunohistochemical analysis
    (2017) Petersen, Morea; Kidson, Susan H
    Wounding of skin initiates a complex series of overlapping cellular events that culminates in the formation of a scar. The negative consequence of most cutaneous scars is the lack of adequate repigmentation of the neoepidermis. In order to effect successful wound healing by restoration of effective skin barrier function, keratinocytes must proliferate and migrate to cover the gap created by the wound in an efficient and timeous manner. Melanocytes are not thought to actively participate in wound repair and closure, since they are primarily responsible for maintenance of normal skin pigmentation. Mechanisms of keratinocyte migration during the re-epithelialisation phase of cutaneous wound healing have been well documented. However, it is not clear how melanocytes contribute to neoepidermis formation and repigmentation. Provision of an optimal epidermal milieu would enable restoration of pigmentation of cutaneous scars for a satisfactory cosmetic outcome. This dissertation contributes to the understanding of melanocyte participation and proliferation during re-epithelialisation of skin using an in vitro skin organ culture model of wound healing. To determine whether the culture model used in this study was reliable, skin explants were cultured over a period of 12 days, fixed and processed for histological analysis. By measuring the lengths of the developing epidermal tongues, it was noted that epiboly was the initial event that occurred at the wound edges. After day two of culture, growth of the epithelial tongues was observed from the wound edges, which peaked at day five. The length of the epidermal tongues remained constant for the remaining culture period up to day twelve, at which stage complete wound closure was observed in some samples. Histological analysis of the tongues revealed that the cultured skin remained viable for the duration of culture period. To establish to what extent keratinocyte proliferation contributed to the growth of the tongues, immunohistochemical staining using the proliferation marker, Ki67, was used. Results show that after an initial lag phase, with no detectable cell proliferation at the wound edges, dividing keratinocytes were seen at day two post-wounding. The number of dividing keratinocytes peaked at day five, where after the numbers remained constant until day twelve of culture. This result supports the validity of the culture model. To uncover whether melanocytes re-enter the neoepidermis during wound during re-epithelialisation, the number of melanocytes per unit of basement membrane was determined using immunohistochemical staining. The melanocyte-specific marker, MART-1/MelanA, was used in conjunction with the proliferation marker, Ki-67, in an optimised dual immunolabelling protocol {Petersen, 2012 #397} to establish whether melanocytes proliferate during re-epithelialisation of wounds. Melanocytes along the basal layer of the epithelial tongues and in the normal epidermis were located and counted. Basal melanocytes (MelanA+) were seen from day two onwards in the normal epidermis and from day five onwards in the developing epidermal tongues. However, at days ten and twelve of the culture period. dividing melanocytes (Ki67+/MelanA+) were seen in the epidermis in locations: proximal to the wound area, at the wound edge and in the developing tongues. This result suggests that melanocyte entry into the wound is delayed until after keratinocyte proliferation has brought about the beginning of neoepidermis formation and growth. This result also demonstrates the proliferation potential of differentiated melanocytes and that de-differentiation of melanocytes can occur under favourable culture conditions as can be seen in this culture model.
  • Thumbnail Image
    Item
    Open Access
    Synthesis and activity of tyrosinase in mouse skin melanocytes
    (1990) Nkabinde, Nkosana Cyril; Kidson, Susan H
    Tyrosinase (E.C. 1.14.18.1) is a key enzyme in the biosynthesis of melanin. The control of melanin sythesis was explored in skin melanocytes of the following strains; wild type (C57BL/6J-C/C) (which maximally synthesize melanin at normal mammalian body temperature, Himalayan (C57BL/6J-cʰ/cʰ) (which maximally synthesize melanin at temperatures below 37°C) and albino (Balb c-c/c) (a mutant which does not synthesize melanin) The effect of a-MSH on tyrosinase activity was initially investigated. A skin culture tyrosinase assay that made it possible to measure the effect of α-MSH on the activity of this enzyme in vitro was first developed. It was found that α-MSH activated the wild type and Himalayan tyrosinase in a dose-dependent manner and that this activation did not require the de novo synthesis of new enzyme. The role of glycosylation on the wild type and particularly the Himalayan tyrosinase activity was next investigated. The results do not support, but are not in conflict with the theory that the Himalayan tyrosinase is inherently underglycosylated. Translation and transcription as additional control mechanisms of tyrosinase activity was finally investigated. The correlation between the levels of tyrosinase activity, abundance of the enzyme and the synthesis of tyrosinase mRNA in wild type, Himalayan and albino mice was determined. It was shown that the levels of newly synthesized tyrosinase and tyrosinase mRNA transcripts were higher in the wild type than in the Himalayan skin. This could account for the reduced tyrosinase activity in the Himalayan mutant at normal body temperature. Low levels of tyrosinase mRNA were found in the albino skin though there was no immunodetectable enzyme in this tissue.
  • Thumbnail Image
    Item
    Open Access
    An ultrastructural and light microscopic study of melanocyte differentiation in chick embryos
    (1991) Stander, Cornelia Steynberg; Kidson, Susan H
    The embryonic source and chemical nature of those factor/s directing the in vivo differentiation of melanocytes from crest cells are as yet unknown. To begin to address this issue, it is important to establish exactly when and where these signa/s first exert their effects. Therefore, in the present study, overtly differentiated melanocytes containing melanin were quantitated in developing Black Australorp X New Hampshire Red chick embryos. In contrast to previous studies, it was found that embryos synthesize melanin from as early as Day 5 of development, and that at this stage, the melanocytes are predominantly dermally located. Between 5 and 8 days, the numbers of both dermal and epidermal melanocytes increase, after which the dermal melanocyte population declines rapidly while the number of epidermal melanocytes continues to increase. These findings suggest that premelanocytes do not have to be epidermally located to initiate terminal differentiation and implicate the dermis as a possible source of melanocyte inducing factor/s. The next step was to examine stages of development prior to the onset of pigment production. For this reason, tyrosinase was purified for use as antigen in the production of a polyclonal antibody. The antibody was tested for specificity by western blotting, - immunocytochemistry and immunoinhibition procedures. Lack of specificity was demonstrated, rendering it unsuitable as an antibody marker for early melanocytes. Fowl melanocytes are thought to differentiate into either eumelanosome- or pheomelanosome synthesizing cells. To test the validity of this concept, embryonic skin of the red/black cross breed were screened for possible mixed type melanocytes by electron microscopy. The melanocytes contained melanosomes with a matrix of irregularly arranged filaments amongst typical eumelanogenic melanosomes. This suggests that these chick melanocytes may synthesize both eumelanosomes and pheomelanosomes in single cells. In a further study on pure breeding New Hampshire Reds, it was found that the melanocytes contained a mixture of typical and less typical pheomelanosomes. Outer membrane indentations in the latter melanosome type suggest that tyrosinase may enter these pheomelanosomes by a mechanism related to that proposed for the melanosomes of goldfish.