Thiol‐Disulfide Status and Acridine Orange Fluorescence of Mammalian Sperm Nuclei

Abstract

ABSTRACT: The relationship between thiol‐disulfide status and acridine orange fluorescence of testicular, epididymal, and ejaculated spermatozoa in several mammalian species was investigated. Spermatozoa were fixed with acetic alcohol, stained with acridine orange, and examined with a fluorescence microscope. The majority of the nuclei of testicular spermatozoa of the hamster, mouse, and rabbit exhibited red acridine orange fluorescence. The proportion of sperm nuclei with red acridine orange fluorescence decreased as the spermatozoa descended the epididymis. Red acridine orange fluorescence was replaced by green acridine orange fluorescence. The site in the epididymis where 100% of the nuclei exhibited green fluorescence was the distal caput in the mouse, the corpus in the rabbit, and the proximal cauda in the hamster. In semen samples from men with proven fertility, normal semen parameters, or both, about 60% to 90% of the nuclei exhibited green acridine orange fluorescence. The proportion of sperm nuclei exhibiting green acridine orange fluorescence was higher in the spermatozoa pellet (containing highly motile spermatozoa) obtained by centrifugation through a Percoll gradient. From experiments using disulfide‐reducing, thiol‐oxidizing and thiol‐detecting agents, we concluded that sperm nuclei fluoresce red when they are treated with acid while their DNA‐associated protamines are poor in disulfides. Under such conditions, DNA is vulnerable to denaturation. Acridine orange binds to denatured (single‐stranded) DNA as aggregates and emits red fluorescence. In contrast, when sperm nuclei are treated with acid while their DNA‐associated protamines are rich in disulfides, DNA is resistant to denaturation. Acridine orange binds to native (double‐stranded) DNA as a monomer and emits green fluorescence. Transformation of sperm nuclei from red‐fluorescing to green‐fluorescing in the epididymis reflects extensive cross‐linking of nuclear protamines by disulfide bonds. In human semen, spermatozoa with red‐fluorescing nuclei are commonly observed. This could be associated with the exceptionally rapid transit of human spermatozoa through the epididymis. Extensive cross‐linking of nuclear protamines is a normal process of sperm maturation. As the resulting stability of the nucleus is apparently important for normal fertilization, assessment of the nuclear integrity by acridine orange fluorescence might be helpful in diagnosing and understanding the causes of infertility in some individuals.