Apart from test systems that directly assess the quality and integrity of the DNA itself, assays have been developed that probe DNA packaging and maturity. This is of particular importance because in spermatozoa, the histones, which are the predominant nuclear proteins in any somatic cell, are replaced during spermiogenesis by protamines in a multistep process. These protamines are disulfide bridge-stabilized, highly basic proteins that fit into the minor grooves of the DNA, neutralize the negative charges of the phosphate groups and thus enable the DNA to form linear arrays fitting into the major groove of the neighboring strand, instead of the voluminous supercoiled ‘solenoids’ present in somatic cells. This results in a highly condensed sperm nucleus in which the DNA takes up about 90% of the total volume. In contrast, the nuclear volume of the DNA in mitotic chromosomes is about 15%, and in somatic cells about 5%.
In the case of disturbed chromatin condensation, histones persist in the sperm nucleus and cause decondensation problems in the male genome after the spermatozoon enters the oocyte.
Thus, patients showing abnormalities of this essential sperm maturation process during spermiogenesis are subfertile or infertile.
Immature, poorly chromatin-condensed sperm nuclei still contain the lysine-rich histones. In an acid–base reaction, acidic aniline blue binds to the basic lysine residues and thus discriminates between lysine-rich histones and arginine/cysteine-rich protamines. This test provides a positive blue staining of spermatozoa with disturbed chromatin condensation, while mature spermatozoa that contain protamines will not be stained.
According to studies by Dadoune et al. and Auger et al., a normal ejaculate should contain at least 75% aniline blue-negative spermatozoa.