Relationship between sperm nuclear protamine free -SH status and susceptibility to DNA denaturation

Data from the sperm chromatin structure assay (SCSA), a flow cytometric measurement of susceptibility of sperm nuclear DNA to denaturation, show strong correlation with the fertility potential of bulls, boars, men and stallions. Previous studies showed a strong relationship between stallion spermatozoa with denatured DNA and the presence of DNA strand breaks. In the present study, the relationship between stallion sperm DNA denaturation and the redox status of -SH groups on the cysteine residues of sperm nuclear protamines that are thought to stabilize chromatin was investigated. Semen samples from 30 stallions were evaluated by the SCSA. Aliquots of the same samples were sonicated to liberate sperm nuclei, purified through a 60% sucrose gradient, stained with an -SH specific fluorochrome (CPM (7-diethylamino-3-(4'-maleimidylphenyl)-4-methyl-coumarin)), and the blue fluorescence of 5000 cells per sample was measured. If S=S bonds stabilize chromatin to inhibit DNA denaturation under the imposed low pH conditions, a low blue intensity would correlate with a low level of DNA denaturation. However, this study showed no correlation (r = -0.199, P = 0.31) of -SH stainability with the extent of DNA denaturation. Thus, other parameters, possibly DNA strand breaks, play a more significant role in susceptibility to DNA denaturation than the extent of S=S bonding within and between protamine molecules. These results also imply that rate of passage through the epididymis may not have significant effects on sperm fertility potential with regard to disulphide bonding status.