Studio del proteoma del seme congelato equino mediante elettroforesi bidimensionale

Proteomics to evaluate the effect of cryopreservation on post-tahw stallion sperm OBJECTIVES AND METHODS The success of cryopreservation in stallion sperm is lower than bull (1). Stallions are commonly selected on the basis of athletic performance records, pedigree and conformation. “Freezability” of semen is a term used to indicate survival rates of sperm populations after a freezing-thaw cycle on the basis of motility parameters. About 20-50% of stallions have sperm with unacceptable freezability (2) and a a lot of variations inter- ad intra stallion also exist, altoughth many different extenders were tested to improve quality of frozen semen. The aim of this study was to compare by proteomic analysis sperm protein expression profile from 6 stallions as studs. They were selected in groups of “high” or “low” freezability quality, according to WBFSH (World Breeding Federation for Sport Horses) guideline ( 35% minimum of PMS, Progressive Motile Spermatozoa). Whole sperm proteins were extracted by 0,5 mL commercial paillettes of frozen semen. After centrifugation cycles to remove extender (INRA82 with abundant skim milk powder declared) protein extracts by 2 commercial paillettes for each stallion were loaded on 7 cm immobilized pH gradient (4-8) IPG strip and separated by IEF (Iso Electro Focusing) as the first dimension, and then with upright SDS-PAGE (polyacrylamide gel electrophoresis) as the second. Every analisys were repeated in double. A usefull method to eliminate extender’s caseine was carried out to avoid interference with semen protein focalisation around pI 4. RESULTS Comparative proteomics analysys of all gels by imagine analysis software showed that expression of 10 protein spots (fig. 1) is related to high or low freezability parameters. Spots 25,27,18 are similar to sp32 (proacrosin binding protein, 28-29kDa, pI 4.8-5.2, teorical 61kDa, 5.9 pI), implicated in acrosomial reaction. In gels appears as a triplette of spots at the same pI, maybe to indicate post-translational modifications. Spot 12 is similar to CRISP3 (Cysteine-rich secretory protein) seminal plasma protein specific to stallion sperm and positively related to fertility (3). Spots 17 and 23 are similar to kallicrein-1E2 (KLK2), protein isoforms, negatively related to prengnancy rate and positively related to ejaculate volume (3). Spots 31 and 32 are highly expressed in “good” freezer stallions, and are compatible with aSFP (acidic protein bovine seminal plasma, 11-12 kDa pI 4.9), a protein of boar seminal plasma spermadhesine, and may play a role against ROS (reactive oxygene species). Spot 68 and 30 can be similar to bovine BSP A1/A2 (16 kDa, pI 4.7-5.5) also called PDC-109, a bull heparin-binding proteins of the seminal plasma positively correlated with good freezeability in this species (4). CONCLUSION Comparative proteomics analysis i this study showed that the expression levels of several proteins are related to high or low semen freezability. Some of that proteins are seminal plasma proteins, and we could hypotithesize that they are involved in protection sperm during cryoconservation and in preservation against oxidative damage. This study also confirme that mere knowledge of the gene sequences is insufficent to elucidate biochemical changes in sperm function, expecially related to post-traslational protein modifications. This study also demonstrate that 2-DE is a crucial step in the workflow of a proteomic approach, and the importance of a correct method to extraction proteins from semen frozen with addicted extender, instead of usual methods based on standard tissues protocols. Further large-scale proteomic studies will lead to the development of novel biomarkers of good semen freezabilty, essential to abtain higher and easier reproductive efficiency and to ansure lower cost and time-loss by breeders.