Proteomic analysis and microtubule dynamicity of human sperm in electromagnetic cryopreservation

Abstract

The proteomic changes, microtubule dynamicity, and quality parameters of human sperm were investigated during cryopreservation in an extremely low electromagnetic field (ELEF) condition. Semen samples were obtained from 210 healthy individuals with normospermia and then were divided into three experimental groups: fresh control, frozen control, and frozen ELEF group. Shotgun proteomics was performed to assess the identification of microtubule proteins of the sperm in experimental groups. Microtubule dynamicity, secondary, and tertiary structure modifications of tubulins, characteristics of transmission electron microscopy of sperm as well as sperm quality parameters were evaluated. The expression ratios of α- and β-tubulins were significantly increased after cryopreservation compared with fresh control while this ratio was not significantly different in frozen ELEF group. The expression ratio of tubulin polymerization-promoting protein was significantly decreased after cryopreservation compared with fresh control. The length, width, and the activity of microtubule, secondary, and tertiary structures of tubulins, motility, and the viability of the sperm were decreased in frozen control as compared with fresh control. The microtubule activity, secondary, and tertiary structures of sperm tubulin in frozen ELEF group were higher than frozen control. Transmission electron microscopy of microtubules showed that the size of the width and length of the microtubules in frozen ELEF group were greater than frozen control. Motility, viability, and reactive oxygen species levels were improved in frozen ELEF group when compared with frozen control. While the microtubule dynamicity of the sperm was affected by the cryopreservation, this trait was improved during the electromagnetic cryopreservation resulted in better motility and viability.

The Application of Ultrasonic Vibration in Human Sperm Cryopreservation as a Novel Method for the Modification of Physicochemical Characteristics of Freezing Media

“…One of the reasons for the increase in the expression of α and β-tubulins might be ascribed to the higher concentrations of these types of tubulins in comparison with other tubulins, elucidating why they are more prone to be affected by environmental conditions such as cold stress. These results are in line with previous studies, indicating irregularities in the expression of α and β- tubulin are increased during cryopreservation of human sperm 30,49 . Since the concentration of α-tubulin is higher than other tubulins, it is more susceptible to damages caused by the freezing process.…”

“…A significant reduction was detected in the presence of the freezing medium containing ULV-exposed water molecules. A large body of evidence indicated the deleterious effects of excessive ROS production on sperm functions 29,30 . Our findings were in line with the results obtained in the study of Sang et al .…”

“…Since the concentration of α-tubulin is higher than other tubulins, it is more susceptible to damages caused by the freezing process. Another reason for an increase in the levels of α- and β-tubulins is microtubule depolymerization during cryopreservation 30 . In this case, α- and β-monomers of tubulins are exposed so that their epitopes are readily available to specific antibodies, and their expression would be clearly detectable in the ELISA method.…”

“…Our data demonstrated that γ-tubulin did not significantly change during cryopreservation in all experimental groups. Perhaps, one of the reasons for these observation is the lower concentration of γ-tubulin in comparison with α and β-tubulin that it is required for microtubule polymerization 30 . Previous reports stated that γ-tubules are resistant to cold stress, and drugs which cause the induction of depolymerization of microtubules 53 .…”

Perspectives in Gamete and Embryo Cryopreservation

“…36 Finally, some authors have proposed the introduction of different cocktails of antioxidants to prevent cryodamage [37][38][39][40][41] or, more recently, the application of a low electromagnetic field. 42 Thawing. Just like freezing, the thawing technique represents a crucial step determining the success of the entire procedure.…”