Could glyoxal be an alternative fixative to paraformaldehyde in human spermatozoa?
Journal of Assisted Reproduction and Genetics, 2026 (SCI-Expanded, Scopus)
- Yayın Türü: Makale / Tam Makale
- Basım Tarihi: 2026
- Doi Numarası: 10.1007/s10815-026-04060-4
- Dergi Adı: Journal of Assisted Reproduction and Genetics
- Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, ATLA Religion Database, BIOSIS, EMBASE, MEDLINE, Academic Search Ultimate (EBSCO), Natural Science Collection (ProQuest), Biological Science Database (ProQuest), Biomedical Reference Collection: Corporate Edition (EBSCO), Health Research Premium Collection (ProQuest), Pharma Collection (ProQuest)
- Anahtar Kelimeler: Fixation of spermatozoon, Glyoxal, Immunoreactivity, Paraformaldehyde
- Yozgat Bozok Üniversitesi Adresli: Evet
Özet
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.