Abstract
Eukaryotic chromatin is organized as radial DNA loops with periodical attachments to an underlying nucleoskeleton known as nuclear matrix. This higher order chromatin organization is revealed upon high salt extraction of cells. To understand the sequential change in the functional organization of chromatin during spermatogenesis, we have analysed the higher order organization of chromatin in different testicular cell types and the epididymal sperm of laboratory mouse. The expansion and contraction of the nucleoid DNA following 2 M NaCl extraction was measured in a fluorescence microscope using ethidium bromide (2.5-200 mg/mL) as an intercalating dye to induce DNA positive supercoils. While the halo size varied among cell types (pachytene DNA most extended, round spermatid least), 5 mg/mL ethidium bromide (EtBr) removed maximum negative supercoils in all the cell types. At higher EtBr concentrations, maximum positive supercoiling occured in pachytene DNA loops. Consistent with this, the pachytene looped domains were maximally sensitive to DNase I, while the elongated spermatids and sperms were highly resistant. Our data suggest that pachytene DNA is in the most open chromatin conformation of all testicular cell types, while round spermatids show the most compact conformation in terms of EtBr intercalation.
Highlights
The ‘ordered arrangement’ of chromatin in the interphase nucleus is maintained by its attachment to the nuclear membrane (Comings, 1968; Comings and Okada, 1970)
Nucleoids were prepared in high salt (2.0 M NaCl) buffer containing different ethidium bromide (EtBr) concentrations (2.5-200 μg/mL)
A halo was not detected in the nucleoids of round spermatids following 2.0 M NaCl extraction, a biphasic change in the diameter of nucleoids was observed as a function of EtBr concentrations (Table 1)
Summary
The ‘ordered arrangement’ of chromatin in the interphase nucleus is maintained by its attachment to the nuclear membrane (Comings, 1968; Comings and Okada, 1970). The 30 nm nucleofilament is further compacted into supercoiled looped DNA domains of 50-100 kb length through attachment to the nuclear matrix (Hancock, 1982; Pienta and Coffey, 1984; VanderWaal et al, 2002, Sheval et al, 2002). The supercoiled loops can be relaxed and visualized as DNA ‘halo’ around nuclear matrix in high salt buffer in the presence of low ethidium bromide (EtBr) concentrations (Benyajati and Worcel, 1976; Vogelstein et al, 1980). It is likely that in response to these variations organizational modifications of chromatin take place in meiotic and post-meiotic cells. The most significant modification is observed during sperm maturation, during which the nucleosomal organization is dismantled in favour of a highly condensed, inert DNA-protein complex, stabilized by disulphide linkages (-S-S-crosslinks) (Ammer and Henschen, 1987)
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