Changes in accessibility of DNA to various fluorochromes during spermatogenesis

Abstract

Accessibility of mouse testicular and vas deferens (vas) sperm cell DNA to acridine orange, propidium iodide, ellipticine, Hoechst 33342, mithramycin, chromomycin A₃, 4′6-dimidino-2-phenylindole (DAPI), and 7-amino-actinomycin D (7-amino-AMD) was determined by flow cytometry. Permealized cells were either stained directly or after pretreatment with 0.06 N HCL. For histone-contanining tetraploid, diploid, and round spermatid cells, HCI extraction of nuclear proteins caused an approximately sixfold increase of 7-amino-AMD stainability but had no signicant effect on DAPI stainability. For these same cell types, the stainability with other intercalating (acridine orange, propidium iodide, ellipticine) and externally binding (Hoechst 33342, mithramycin, chromomycin A3) dyes was increased by 1.6- to 4.0-fold after HCI treatment. In sharp contrast, HCI treatment of vas sperm did not increase the staining level of 7-amino-AMD, DAPI, or propidium iodide but did increase the staining level for the other intercalating dyes (1.3- to 1.5-fold) and external dyes (1.3- to 1.9-fold). Elongated spermatids that contain a mixture of protein types including histones, transition proteins, and protamines demonstrated the greatest variability of staining with respect to type of stain and effect of acid extraction of proteins. In general, for nearly all dyes, the round spermatids had an increased level and tetraploid cells had a decreased level of stainability relative to the same unit DNA content of diploid cells. The observed differential staining is discussed in the context of chromatin alterations related to the unique events of meiosis and protein displacement and replacement during sperm differentiation.