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 commonly associated with cases of male factor infertility.
To better understand the process of sperm nuclear condensation and maturation, we isolated sperm nuclei from cells with markers of good and poor compaction from an ejaculate using a density gradient. 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. We have also demonstrated that the number of cells in which we are able to detect TOP2A by immunocytochemisty is higher in spermatozoa with poor chromatin compaction. 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.