Unravelling subcellular and ultrastructural changes during vitrification of human spermatozoa: effect of mitochondria-targeted antioxidant and permeable cryoprotectant

The aims of the study were to improve sperm vitrification by using mitochondria-targeted antioxidants (Mito Q), reveal ultrastructural changes in spermatozoa due to the use of permeable cryoprotectants, and report alteration of functional proteins during the sperm vitrification process. For this, each of 20 swim-up-prepared ejaculates was divided into four groups : control, treatment 1, 2 and 3. We performed label-free quantitative proteomics and identified 1759 proteins, of which 68, 60, 90, and 81 proteins were altered in Treatment 1 (0.02µM Mito Q), Treatment 2 (1 % glycerol), and Treatment 3 (Mito-Glycerol groups) respectively compared to control (Fresh sperm). In particular, actin proteins were not affected during sperm vitrification but few actin-associated proteins were altered during the vitrification process. Interestingly, tubulins and outer dense fiber proteins were not affected during the vitrification process. Some of the identified ubiquitinating enzymes were affected during sperm vitrification but deubiquitinating enzymes were not affected during the vitrification process. Several proteins were identified for phosphorylation and dephosphorylation but only a few are altered during the sperm vitrification process. Interestingly, twenty-seven heat shock proteins were identified but only one is altered in the control. Similarly, several candidate proteins involved in sperm-egg fusion and fertilization (IZUMO1, Tektin, etc) were identified but not affected during the vitrification process. In conclusion, we demonstrated that MitoQ attenuates vitrification-induced ultrastructural changes and alteration in key proteins involved in sperm functions and fertilization.