Analysis of topological organization of chromatin during spermatogenesis in mouse testis

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

Analysis of DNA looped domains organization duringTriturus cristatusspermatogenesis

“…The biphasic changes of the nucleoid and halo sizes of testicular cells in response to increasing concentrations of EtBr show that, like many other cell types studied (Vogelstein et al ., 1980, Risley et al ., 1986, Narayan & Raman, 2004), DNA of newt germinal cells is organized in negatively supercoiled domains. A remarkable finding is the similar biphasic modulation of mature spermatozoids of epididymes but on a much smaller scale, which would lead to the assumption that the nucleosome organization is not completely lost in these highly differentiated cells.…”

“…The apparent lack of looped domains is the consequence of attachment of an even greater number of regions to the nuclear matrix. It is appropriate to assay the sensitivity of histone-depleted and EtBr-relaxed chromatin, as DNase I sensitivity could be a function of the local displacement of histones to form a nucleosome-free region of chromatin (Narayan & Raman, 2004). DNase I does not cleave randomly on the loop: the region closest to the matrix may be less sensitive than the most distant region (Narayan & Raman, 2004).…”

“…In present study, starting from Narayan & Raman investigations on topological organization of chromatin during mouse spermatogenesis (2004), the higher order chromatin organization in Triturus cristatus spermatogenesis has been analysed.…”