Sperm Chromatin Status and DNA Fragmentation in Mouse Species with Divergent Mating Systems

Abstract

Sperm DNA integrity and chromatin status serve as pivotal indicators of sperm quality, given their intricate link to sperm function, embryo development, and overall fertility. Defects in chromatin compaction, which are often associated with compromised protamine content, can lead to damaged DNA strands. In this study, the chromatin status and possible correlation with DNA damage was assessed in males of three mouse species: Mus musculus, M. spretus, and M. spicilegus. We employed various staining methods, including aniline blue, methylene blue (Diff-Quik), toluidine blue, and chromomycin A3, to assess chromatin compaction in cauda epididymal sperm. Samples were also analyzed by the sperm chromatin structure assay (SCSA) to estimate DNA fragmentation (%tDFI, %HDS). Analyses were carried out on freshly collected sperm and cells incubated for 3 h in a HEPES-buffered modified Tyrode's medium simulating conditions of the female reproductive tract. Notably, the analysis of chromatin status yielded minimal abnormal values across all three species employing diverse methodologies. SCSA analyses revealed distinct variations in %tDFI between species. Following sperm incubation, the percentages of sperm stained with methylene blue exhibited differences among the species and were significantly correlated to the DNA fragmentation index. HDS demonstrated correlations with the percentages of sperm stained by aniline blue, methylene blue, and chromomycin A3. Overall, chromatin compaction was high across all species, with limited differences among them. The relationship between chromatin status and DNA integrity appeared to be related to levels of sperm competition among species.

Keywords: DNA fragmentation; Diff-Quik; SCSA; aniline; chromatin compaction; chromomycin A3.

Figure 1

Spermatozoa from Mus spretus stained with (A) aniline blue, a: spermatozoon with mature chromatin (light blue or pink), b: spermatozoon with immature chromatin (dark); (B) Diff-Quik, a: spermatozoa with normally compacted chromatin (light violet), b: spermatozoon with abnormally compacted chromatin (dark violet); (C) toluidine blue, a: spermatozoa with normal chromatin (light blue), b: spermatozoa with damaged chromatin (dark blue/purple); (D) chromomycin A3, a: spermatozoa with correct protamination (dull green), b: spermatozoon with incorrect protamination and completely stained head (bright green).

Figure 2

Relationships between DNA damage and chromatin status in freshly collected sperm and after 3 h of incubation in mT-H medium. Linear regressions between values of %HDS and % positive cells in each staining assay are plotted for all the species (n = 5 for each species). (A) Aniline blue in fresh sperm (0 h). (B) Aniline blue after 3 h of incubation. (C) Aniline blue Δ3 h–0 h. (D) Diff-Quik in fresh sperm (0 h). (E) Diff-Quik after 3 h of incubation. (F) Diff-Quik Δ3 h–0 h. (G) CMA3 in fresh sperm (0 h). (H) CMA3 in sperm after 3 h of incubation. (I) CMA3 Δ3 h–0 h. Values with p < 0.05 were considered statistically significant (in bold). Abbreviations. AB: aniline blue, DQ: Diff-Quik, CMA3: chromomycin A3, HDS: high DNA stainability.