Spermatogenesis is an extremely specialised process that generates a cell capable of the protection and delivery of the paternal genome to the oocyte. During the development of a spermatozoon, the basic chromatin structure of DNA bound to histones is drastically altered, and nuclear volume is greatly reduced. Importantly poor chromatin compaction has been associated with interruption of the histone/protamine 1/protamine 2 ratios in infertile men, however it has been shown these ratios are subject to change over time and to environmental stresses.
To better understand the process of sperm nuclear condensation, we isolated the sperm nuclei from cells with markers of good and poor compaction from an ejaculate. Comparative proteomics was performed on the nuclear proteins, using the quantitative SWATH platform on the Sciex 6600 Triple ToF. We confidently identified 342 proteins, and of these proteins 20 were found to be more abundant in the sperm possessing poor chromatin compaction, many of which are associated with nucleoplasm. Immunoblots using an antibody against TOP2A and PDIA3 confirmed the proteomic analysis. Unexpectedly, no changes were observed in any of the identified histone peptides (H4, H3.3, H1T, H2A/B), nor for protamine 2. Our data suggests an alternate explanation for poor chromatin compaction. Rather than changes in histone or protamine content, it appears that retained or excess nucleoplasm is more prevalent in poorly compacted nuclei.