The influence of fixation on the relative amount of cytoplasmic ribosomes in mouse epidermal basal keratinocytes
- 22 Downloads
The nature and significance of so-called dark keratinocytes in the epidermis during chemical carcinogenesis is still a matter of concern and debate. Based on ultrastructural observations it has been suggested that dark cells most often are shrunken cells. Reports on skin carcinogenesis, however, claim that dark cells are a sign of ongoing tumor promotion and represent those stem cells in the epidermis from which the tumors originate. It is therefore important to find out whether these cells are simply injured and shrunken cells, or vital cells of great importance for carcinogenesis. Dark cells are assumed to be rich in ribosomes. There is evidence, however, that the observed number of dark cells is highly dependent on tissue fixation. In the present ultrastructural study, morphometric methods were used to compare the effects of two different fixation procedures on the amount of cytoplasmic ribosomes in dark cells from both untreated and carcinogen-treated hairless mouse epidermis. The results show that the ultrastructural features of both dark and clear cells vary considerably with different fixation procedures. In acetone-treated controls typical dark cells are only observed when the fixative has a lower osmotic activity than the plasma. With iso-osmolal fixation typical dark cells are not observed. After an abortive two-stage carcinogenesis treatment, in which a single application of 9,10-dimethyl-l,2-benzanthracene (DMBA) in acetone was followed by a single application of 12-O-tetradecanoyl-13-acetate (TPA) in acetone, signs of cell injury could be found after both fixation procedures. With DMBA/TPA and hypo-osmolal fixation the number of dark cells seemed to increase, whereas only signs of cell injury with occurrence of some heavily altered “clear cells” dominated the picture with iso-osmolal fixation. Morphometry showed that both the numerical and the volumetric densities of cytoplasmic ribosomes in basal keratinocytes varied most significantly with the fixation procedure used. The cytoplasmic volumes did not vary in a way that could explain these differences. One might therefore assume that the number of ribosomes depends on the fixative. Large swelling artifacts occurred when a fixative with low osmotic activity was used, leading to compression of neighboring cells. Hence, an increased ribosomal density reported previously in dark cells is probably related to such cell volume artifacts and does not reflect an actually increased quantity of ribosomes. With both fixation procedures, a single application of DMBA followed by one of TPA appeared to produce an increased number of ribosomes in basal keratinocytes. When hypo-osmolal fixation was used, however, treatment with DMBA/TPA did not influence the cytoplasmic volume or the numerical density of ribosomes, in dark cells. This might indicate that so-called dark keratinocytes following DMBA/TPA treatment are functionally inactive cells that appear more vulnerable than active cells to compression during hypo-osmolal fixation.
Key wordsDark cells Microscopy electron Morphometry Mouse epidermis Ribosomes
Unable to display preview. Download preview PDF.
- Falck B, Andersson A, Elofsson R, Sjøborg S (1981) New views on epidermis and its Langerhans cells in the normal state and in contact dermatitis. Acta Derm Venereol (Stockh) [Suppl 99] 61:3–27Google Scholar
- Ghadially FN (1986) Diagnostic electron microscopy of tumours, 2nd edn. Butterworths, London, pp 326–333Google Scholar
- Gundersen HJG (1977) Notes on the estimation of the numerical density of arbitrary profiles: the edge effect. J Microsc 111:219–223Google Scholar
- Hayat MA (1981) Fixation for electron microscopy. Academic Press, New York, pp 9–261Google Scholar
- Hume WJ (1987) A pathologist’s view of cell death. In: Potten C S (ed) Perspectives on mammalian cell death. Oxford University Press, Oxford, pp 73–79Google Scholar
- Iversen U, Iversen OH (1967) Cycles of hair growth in hairless mice. Acta Path Microbiol Scand 69:50–62Google Scholar
- Kerr JFR, Searle J, Harmon BJ, Bishop CJ (1987) Apoptosis. In: Potten C S (ed) Perspectives on mammalian cell death. Oxford University Press, Oxford, pp 93–128Google Scholar
- Snedecor GW (1964) Statistical methods. The Iowa State University Press, Ames, pp 329–393Google Scholar
- Takigawa M, Komura J, Ofuji S (1978) Early fine structural changes in human epidermis following application of croton oil. Acta Derm Venereol (Stockh) 58:31–35Google Scholar
- Thrane EV, Huitfeldt HS, Clausen OPF, Roop D, Yuspa SH (1988) Differentiation and proliferation kinetics of normal and TPA-treated mouse epidermis. Proceedings of the 24th Meeting of the Norwegian Biochemical Society. Lobo Grafisk A/S, Oslo, p 93Google Scholar
- Williams MA (1977) Quantitative methods in biology. In: Glauert AM (ed) Practical methods in electron microscopy, vol 6. Elsevier/North-Holland, Amsterdam, p 68Google Scholar