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

Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusion: 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.