Neutral lipids accumulate in cellular lipid droplets. These droplets vary remarkably in number and amount between cells. In the present studies, the variability in lipid content was quantified by comparing the coefficient of variation of fluorescence histograms of nile red lipid-stained cells to the variability of cell size or cell protein distributions. This measure of lipid droplet variability persisted through a wide range of cell lipid content and averaged 4.4-fold more variability than cell size and 2.6-fold more variability than cell protein content. While looking for possible explanations for this variability, it was determined that cell to cell variability could not be explained by multiple clonal populations of cells or the confluence of the cell monolayer. Analysis of lipid variability using a more droplet-specific fluorescent dye, bodipy, reduced variability by about 44%. Cell cycle analysis revealed that G2 + M cells contained more lipid than S-phase cells, which in turn contained more lipid than Go + GI cells, but that variability was equally large throughout the cell cycle. The cholesteryl ester hydrolase inhibitor, diethylumbelliferyl phosphate, inhibited hydrolysis of both cholesteryl esters and triglycerides. Lipid content of diethylumbelliferyl phosphate-treated cells increased while the variability in lipid staining decreased by an average of 72%. Thus, the excess lipid fluorescence variability compared to cell size or protein fluorescence could in part be explained by variability in cellular hydrolysis of triglyceride and cholesteryl ester. Excess lipid fluorescence variability could be reduced by an average of 44% when a more lipid droplet-specific stain was used instead of nile red. Key terms: Nile red, fluorescence, quantitative lipid staining, bodipy, diethylumbelliferyl phosphate, lipid dropletsThe mechanisms leading to lipid accumulation in cells are not well understood. Since lipid accumulation in one cell type, the macrophage, may act as the initiating event in the atherosclerotic process (2,19), considerable effort has been directed toward understanding this process. The lipophilic dye nile red has been used in some studies of macrophages (11,131. These studies suggest that nile red might act as a quantitative lipid stain since nile red fluorescence is increased in at least rough proportion to the amount of lipid fed to the macrophages (13). Compared to the variability of protein fluorescence in most cells (81, nile red fluorescence seems quite variable. The present studies were prompted by these two observations.The MA-10 cells are a clonal strain of Leydig tumor cells. These cells have been utilized extensively by this laboratory for studies of cholesterol metabolism (61, storage (4), and transport (5,15,16). Unlike macrophages, MA-10 cells are clonally selected and thus should represent a single genetic population of cells. This characteristic should prove useful in studies exploring the nature of lipid droplet variability. The MA-10 cells can also be manipulated in a manner lendi...

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The aim of these experiments was to investigate the expression of cyclin D1 and of oestradiol receptors as well as the level of [(3)H]oestradiol binding in leiomyoma and adjacent myometrium from human uteri at different menstrual phases and at an early stage of menopause. [(3)H]oestradiol binding was determined by saturation analysis, while the oestradiol receptor (ER) alpha and beta and cyclin D1 levels were determined by Western blot analysis of 16 samples of human leiomyomas and corresponding myometria at different hormonal stages. In leiomyomas during all phases of the menstrual cycle, ERalpha expression, high affinity oestradiol binding and cyclin D1 expression were all elevated in comparison with adjacent myometrium. ERbeta expression and low affinity oestradiol binding were enhanced in leiomyomas only during the proliferative phase. During menopause, ERbeta expression and low affinity binding were enhanced in leiomyomas, while the ERalpha expression was not significantly enhanced and cyclin D1 levels were similar to that in myometrium. Only the oestradiol binding exhibited any menstrual cycle-related changes. Our data suggest the involvement of cyclin D1 in the growth of leiomyomas during the menstrual cycle. In menopause, there appears to be a switch from ERalpha to ERbeta expression in leiomyomas, and the induction of cyclin D1 is decreased. The regression of tumour may ensue from these changes at menopause.