The aim of this study is to determine why, in contrast to expectations based on the Lyon hypothesis, a variable number of nuclei of cells from mammalian females are sex chromatin negative. The frequency of sex chromatin positive nuclei was determined in cell cultures of varying cell densities. The cells were derived from seven chromosomally normal human female embryos, one newborn female with an extra E group chromosome and two normal male embryos. In all cultures of females the frequency of sex chromatin positive nuclei increased linearly from about 35% to 60% at cell densities of less than one cell per 0.01 mm2 of culture surface to 90% to 100% at densities of 20 to 125 cells per 0.01 mm2. This frequency-to-density relationship was independent of the mitotic rate and the rate at which cell density increased. When large variations in cell density were produced intentionally on the same glass coverslip, sex chromatin frequency was related to the density of cells in any one area of a coverslip and seemed to be largely independent of the cell density in other parts of the coverslip. The frequency of sex-chromatin-like bodies of male cultures remained very low at all cell densities. These and other preliminary observations described suggest that, in the nucleus of the female, sex chromatin formation resulting from the condensation of an X chromosome at interphase is not directly related to the mitotic cycle but may be related to the metabolic state of the cell.

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