7‐aminoactinomycin D binding and the final stages of sperm chromatin processing in the mouse

Quantitative fluorescence microscopy is used to compare the binding of the fluorescent, 7‐amino derivative of actinomycin D to a sonication resistant fraction of late‐step mouse spermatids and sperm isolated from three regions of the epididymis. Staining conditions are described that optimize 7‐AMD binding to air‐dried smears of these cells and permit quantitative analyses of 7‐AMD binding to sperm chromatin at the single cell level. These studies show that the step 12–16 spermatid undergoes an 8–10‐fold reduction in 7‐AMD binding as it completes differentiation in the testis and progresses through the epididymis. The first 2–3‐fold reduction in binding, associated with biochemical changes in chromatin structure brought about during spermatid differentiation, occurs gradually and is complete by the time the sperm reaches the caput epididymis. An additional four‐fold reduction is observed as the sperm passes through the corpus. This final alteration, which occurs in a single concerted step, may be reversed by dithiothreitol treatment and appears to be effected through the formation of a final, small group of sperm nuclear protein disulfide crosslinks.