A comparative study of spermatozoal chromatin using acridine orange staining and flow cytometry

Spermatozoa obtained from fish (Clarias gariepinus), human (Homo sapiens), turkeys (Meleagris gallapova), rats (Rattus norvegicus), hamsters (Mesocricetus auratus), and monkeys (Macaca fascicularis) were stained with acridine orange before measuring fluorescence by flow cytometry. These mature sperm from various species produced different intensities of fluorescence while displaying similar ratios of red/green fluorescence. Comparison of the green fluorescence values for the various species showed the sequence (descending order of fluorescence values) human, turkey, monkey, hamster, rat and fish. The DNA complement (as base pairs in the haploid genome) of the various species did not increase in direct proportion to the fluorescence values. This suggests that the DNA was not equally accessible to the dye in the different species tested. The similarity in ratios of red/green fluorescence suggests that the structure of DNA in the chromatin is similar in the different species but abnormal 'satellite' populations of cells that show higher red/green fluorescence ratios than the parent population have been found in sperm samples from monkeys and from some infertile men. Their high red fluorescence intensities were not caused by RNA because treatment with RNAse did not alter the red fluorescence. It is possible that these cells contain larger amounts of denatured (single stranded) DNA.