Flow cytometry of DNA in mouse sperm and testis nuclei.

Mutations that occur in spermatogenic cells may be expressed as changes in DNA content, but developmentally-dependent alteration of its staining properties complicates the quantitation fo DNA in individual germ cells. These alterations have been studied with flow cytometric techniques. Nuclei from mouse testis cells and sperm were stained by the acriflavine-Feulgen method. The fluorescence intensity frequency distribution of nuclei of testis cells was characterized by 2 major and 5 minor peaks. Nuclei sorted from the various peaks with a fluorescence-activated cell sorter were identified microscopically. These data were confirmed by generation of peaks with nuclei prepared from cell suspensions enriched in specific cell types. One of the major peaks corresponded to round spermatid nuclei. The other major peak, located at 0.6 of the fluorescence intensity of the round nuclei, corresponded to elongated spermatid nuclei. Purified nuclei of epididymal and vas deferens spermatozoa displayed asymmetric fluorescence distributions. A minor peak at 0.8 the intensity of the round spermatid nuclei was tentatively assigned to elongating spermatids. 2 of the minor peaks, located at 1.7 and 2.0 times the fluorescence intensity of the round nuclei, corresponded to clumps of 2 haploid and diploid nuclei. The additional peaks, located at 3.0 and 3.7 times the fluorescence intensity of round spermatid nuclei correspond to leptotene and zygotene spermatocytes and to late pachytene spermatocytes, respectively. These peaks contained clumps of nuclei. The homogeneity of the nuclei sorted from the peaks, as well as the relative sizes of the peaks, was enhanced when the nuclei were prepared from cells enriched in specific stages of development. The relative fluorescence intensities of the various testis nuclei were characteristic and repeatedly found but were not stoichiometric with the DNA content of the nuclei.