Resumen de Freezability markers for boar sperm: new proteomic approaches
Sperm cryopreservation is currently the most suitable technique to store boar sperm samples for a long period of time. This method has many advantages, including preservation of specific genetic material from breeds and lineages, planning of reproductive trials in breeding farms and sanitary control of samples. However, this technique also has limitations that heavily rely upon the cell sensitivity to cold shock, very high in the case of boar sperm. In addition, not all boar ejaculations present the same freezability (i.e. resilience to withstand cryopreservation), but rather a great variation between and within boars exists. This is related to the sperm resistance to cold shock and allows the classification of boars and their ejaculations into good (GFE) and poor (PFE) freezability boars/ejaculates. In this context, the present Thesis aims to gain new insights into the evaluation and prediction of sperm freezability, thereby preventing PFE cryopreservation. With this purpose, three different objectives were set and addressed by separate, but complementary studies. The first aim was to determine, through Western blot, whether small heat shock protein 10 (ODF1/HSPB10) and voltage-dependent anion channel 2 (VDAC2) were reliable freezability markers. The second objective was to find potential freezability markers from comparing the sperm proteome of GFE and PFE through two-dimensional difference gel electrophoresis (2D-DIGE). The third aim was to compare the fresh seminal plasma proteome of GFE and PFE through 2D-DIGE, again seeking potential markers for boar sperm freezability. Results from 2D-DIGE obtained were in all cases validated by Western Blot. For the first two objectives, 26 and 34 boar ejaculations, respectively, were split into two fractions, one for protein determination and the other for cryopreservation purposes. Ejaculations were subsequently classified into two groups (GFE and PFE) using progressive motility and sperm viability at 30 and 240 minutes post-thawing. In the first study, VDAC2 amounts in fresh sperm were found to be significantly higher in GFE than in PFE. In contrast, ODF1/HSPB10 did not significantly differ between groups. Furthermore, principal component and multiple regression analyses showed the component explaining the 78.41% of the variance in ejaculate freezability at 240 min post-thawing was significantly correlated with VDAC2 amounts, demonstrating that this protein may be used to predict boar sperm freezability before cryopreservation procedures take place. As far as the second objective is concerned, 2D-DIGE allow to identify two potential freezability markers in boar fresh sperm: acrosin binding protein (ACRBP) and triosephosphate isomerase (TPI). After validation through Western Blot, ACRBP content was significantly higher in GFE than in PFE, whereas TPI amounts were significantly lower in the former that in the latter. Pearson’s linear correlations were used to confirm the association of these two proteins with post-thaw sperm viability and motility For the third objective, 18 boar ejaculations were classified as GFE and PFE (as in the other studies) and fresh seminal plasma proteomes were compared, Following 2D-DIGE, two different proteins (fibronectin-1, FN1, and glutathione peroxidase 5, GPX5) were found to significantly differ between GFE and PFE. However, after Western Blot validation and correlation analysis FN1 but not GPX5 was found to be a reliable marker of boar sperm freezability. Therefore, the main conclusion of this Dissertation is that VDAC2, ACRBP and TPI in fresh sperm and FN1 is fresh seminal plasma may be used to predict the sperm resilience to withstand cryopreservation procedures. Taking together, these findings provide new insights into molecules involved in boar sperm freezability shedding light on the mechanisms involved in boar sperm cryotolerance. Furthermore, they may help to develop new tests to detect sperm freezability capacity at molecular level avoiding the costs and time on cryopreserving PFE. This could ultimately lead the swine industry to reconsider its interest for cryopreserved sperm doses.
