Persistent histones in immature sperm are associated with DNA fragmentation and affect paternal contribution of sperm: A study of aniline blue staining, fluorescence in situ hybridization (FISH) and DNA nick translation

Abstract

We have tested whether aniline blue staining of sperm is related to DNA chain breakage. During sperm development there are concurrent nuclear and cytoplasmic processes, including meiosis, DNA repair, histone-protamine replacement, and cytoplasmic extrusion. Aniline blue, a marker of persistent histones in immature sperm, may stain sperm light (L, mature sperm), intermediate (IN) and dark (D, immature sperm, high level of persistent histones). It was reported that the immature D sperm do not show FISH chromosome labeling, whereas in L and IN there are well characterized FISH signals. We have examined, with a combination of aniline blue staining and FISH or aniline blue staining and DNA nick translation, whether the lack of FISH signals in D sperm is caused by an interference of aniline blue staining with FISH, or if in immature D sperm there is also DNA fragmentation, thus the FISH probes fail to recognize the chromosomes. Sperm were stained with aniline blue, and the microscopic fields of aniline blue stained sperm were digitized and captured by the computer assisted Metamorph program (Universal Imaging Co., PA). Subsequently, the aniline blue stain was bleached with 30% methanol-DDW, and the slides were subjected to either multicolor FISH using X, Y, and 17 chromosome probes, or to DNA nick translation. Nick translation generates color via enzyme linked nucleotides inserted at DNA chain breaks. The color intensity is proportional with the extent of DNA fragmentation, and graded as light nick (LN), intermediate nick (IMN) and dark nick (DN) sperm. Further, the saved aniline blue stained sperm images were compared with those of the same sperm after FISH or DNA nick translation treatment. Statistical evaluation by Student t, Mann-Whitney, and the Spearman’s correlation tests was carried out by the SigmaStat program (Jandel, CA). We confirmed that D sperm indeed fail to show FISH signal, whereas in L and IN sperm the fluorescence chromosomes are well detectable. The sperm stained for aniline blue and nick translation provided evidence for extensive DNA degradation in D sperm. In 5 men we studied 600 spermatozoa. There was an agreement between the staining patterns with aniline blue and subsequent nick translation in 82.8% of sperm (500 of 600): 46.5% of sperm stained as L and LN, 27.7% of sperm stained IM and IMN, and 7.6% of sperm stained D and DN, respectively. Thus, sperm with high level of persistent histones have also shown extensive DNA degradation. Only 10 sperm of 600 had an L-DN or D-LN discordant pattern. The correlation between the aniline blue and nick translation staining patterns was r=0.82, p<0.001. Using probes for persistent histones and fragmented DNA in the same sperm indicates that diminished maturity sperm show defects in both histone-protamine replacement and DNA fragmentation. Both persistent histones and DNA fragmentation affect the paternal contributions of sperm to the zygote. Thus, aniline blue staining may facilitate the quick and inexpensive prospective evaluation of DNA integrity in husbands treated with assisted reproduction.