Unraveling Subcellular and Ultrastructural Changes During Vitrification of Human Spermatozoa: Effect of a Mitochondria-Targeted Antioxidant and a Permeable Cryoprotectant

Abstract

Mitochondria-targeted antioxidants have great potential to counterbalance the generated reactive oxygen species (ROS) because they cross the inner membrane of the mitochondria. Still, their use was not reported in vitrified human spermatozoa. Our laboratory has successfully vitrified spermatozoa without the use of permeable cryoprotectants, but subcellular-level evidence was missing. Therefore, this study aimed to improve spermatozoa vitrification using a mitochondria-targeted antioxidant (mitoquinone, MitoQ), reveal ultrastructural changes in the spermatozoa due to the use of a permeable cryoprotectant, and report alterations of functional proteins during the spermatozoa vitrification process. For this, each of 20 swim-up-prepared ejaculates was divided into seven aliquots and diluted with a vitrification medium supplemented with varying concentrations of MitoQ (0.02 and 0.2 μM), glycerol (1, 4, and 6%), and a combination of MitoQ and glycerol. All aliquots were vitrified by the aseptic capillary method developed in our laboratory. The spermatozoa function assays revealed that the addition of either MitoQ (0.02 μM), glycerol (1%), or a combination of MitoQ (0.02 μM) and glycerol (1%) in the vitrification medium results in better or equivalent spermatozoa quality relative to the control. Transmission electron microscopy revealed that MitoQ protects the spermatozoa from undergoing ultrastructural alterations, but glycerol induced ultrastructural alterations during the vitrification process. Next, we performed label-free quantitative proteomics and identified 1,759 proteins, of which 69, 60, 90, and 81 were altered in the basal medium, 0.02 μM MitoQ, 1% glycerol, and Mito-glycerol groups, respectively. Actin, tubulins, and outer dense fiber proteins were not affected during the vitrification process. Some of the identified ubiquitinating enzymes were affected during spermatozoa vitrification. Only a few proteins responsible for phosphorylation were altered during vitrification. Similarly, several proteins involved in spermatozoa–egg fusion and fertilization (IZUMO1 and Tektin) were not affected during the vitrification process. In conclusion, MitoQ attenuates the vitrification-induced ultrastructural changes and alterations in the key proteins involved in spermatozoa functions and fertilization.

Cryoprotectants-Free Vitrification and Conventional Freezing of Human Spermatozoa: A Comparative Transcript Profiling

“…However, some recent literature indicates that mature spermatozoa can use mitochondrial RNA transcripts for protein translation during the final maturation steps before fertilization [ 13 , 14 ]. In our previous study, we found that some proteins are altered, resulting in higher or lower expression levels of proteins in spermatozoa subjected to vitrification; due to proteomic study only, we can propose that some functional proteins are not altered/modified that were reported to be altered in most of the conventional slow freezing method studies [ 15 ]. Like our studies, the increased or decreased alteration of some proteins in cryopreserved spermatozoa has been reported in some studies [ 16 , 17 , 18 ].…”

The relationship between reactive oxygen species, DNA fragmentation, and sperm parameters in human sperm using simplified sucrose vitrification with or without triple antioxidant supplementation

“…Our results revealed significantly lower sperm viability and motility in the vitrified groups than in the fresh control group, which is in agreement with earlier studies [ 29 - 31 ]. Cryopreservation has deleterious effects on sperm motility by damaging the plasma membrane and mitochondrial function [ 29 , 32 ]. However, we did not find statistically significant differences in the viability and motility parameters of post-thawed samples between sucrose vitrification and the commercial sperm freezing medium.…”

“…This result is consistent with other research on human sperm cryoinjury [ 39 ], which indicated that the extracellular ROS levels increased during cryopreservation by increasing mitochondrial and membrane NADPH oxidase-5 (NOX5) activity. The increased ROS production following cryopreservation may be a result of mechanical damage to the sperm plasma membrane [ 32 , 40 ]. An alternative explanation is that the balance between ROS production and antioxidant scavenging systems is disrupted during the freezing-thawing process.…”

Epigenetic Alterations in Cryopreserved Human Spermatozoa: Suspected Potential Functional Defects

“…Cryopreservation involves sudden osmotic and temperature changes that may lead to molecular disturbances in the sperm transcriptome and proteomics [7][8][9]. A thorough understanding of these molecular disorders during cryopreservation is essential in order to optimize current cryopreservation protocols [2].…”