Application of the New Stereological Probes to the Study of the Melanosome in Cloudman S91 Melanoma Cells

The relationship between melanosome size and number and melanin content has been investigated in Cloudman S91 melanoma cells growing in vitro using both "model-based" and "design-based" stereological procedures. Cells were cultured for 4 days, harvested at daily intervals, and resin-embedded for light and electron microscopy; one aliquot of each sample of cells was assayed to determine its melanin content. By comparing their volume-weighted mean nuclear volume and their number-weighted mean nuclear volume, we have found that the nuclei of Cloudman melanoma cells form a fairly homogeneous population. The volume fraction and absolute volume of premelanosomes (V_Vpm,cell and V_pm) and mature melanosomes (V_{Vmm, cell} and V_mm) were all found to decrease progressively throughout the period of culture as did the number of premelanosomes (N_pm) and mature melanosomes (N_mm). Whilst the volume-weighted mean volume of individual stage I and stage II premelanosomes, (V̄_Vipm) remained fairly constant at about 10 nm³, the volume of individual stage III and IV mature melanosomes showed significant variation ranging between about 13 nm³ and 32 nm³. The melanin content of the cells decreased progressively over the 4 days of culture. There were, however, considerable variations in both the average melanin content per unit volume of mature melanosomes, in the range 170-600 fg/μm³, and in the melanin content per individual mature melanosome, in the range 3-12 fg. Our findings show that stereological techniques can provide unbiased and sensitive tools for the study of the morphological basis of melanogenesis; their value will become even more evident when they are combined with techniques for the localization of melanogenic enzymes and their substrates.