Could glyoxal be an alternative fixative to paraformaldehyde in human spermatozoa?

Our previous studies demonstrated the superiority of paraformaldehyde (PFA) over glyoxal (Gly) fixation in stem cells, oocytes, and embryos. Here, we compared these two aldehyde-based fixatives in human spermatozoa to determine whether Gly, which is reported to better preserve immunoreactivity, can provide effective and consistent fixation comparable to PFA.

The fixation performance of PFA (3.5%) and Gly (3%), with or without 0.01% Triton-X (TX) permeabilization, was evaluated side-by-side in live and fixed human spermatozoa. Analyses included total protein quantification by Western blotting, epitope detection by super-resolution confocal laser scanning microscopy, ultrastructural assessment by scanning electron microscopy, and DNA fragmentation analysis by TUNEL assay coupled with flow cytometry.

Protein fixation assays demonstrated that PFA was superior to Gly in preserving total cellular proteins. Similarly, the sperm-specific membrane protein IZUMO1 was better retained after PFA fixation. In contrast, Gly combined with subsequent TX permeabilization provided superior labeling of nuclear DNA, the acrosomal protein ACRBP, a nuclear DNA-methylating enzyme DNMT3A and TUNEL-based detection of DNA fragmentation compared with all PFA-based fixation conditions. No significant differences between fixatives were observed in ultrastructural preservation, post-fixation live mitochondrial staining.

PFA was more effective for preserving total and membrane-associated proteins in human spermatozoa, whereas Gly represented a promising and less toxic alternative for studies targeting nuclear components and DNA-associated molecules. These findings indicate that the choice of fixative should be determined according to the specific cellular targets under investigation, particularly cytoplasmic, membrane-associated, or nuclear proteins and enzymes.