Localización, aislamiento y caracterización de proteínas tiólicas durante la maduración del espermatozoide

CABRILLANA M.E; MONCLUS MA; VINCENTI AE; FORNÉS MW; YUNES R

Huerta Grande, Córdoba

Congreso; I Reunión Conjunta de Sociedades de Biología de la República Argentina; 2007

Sociedades de Biología de la Republica Argentina, Cuyo, Córdoba, Tucumán, rosario y Bs. As.

LOCATION, ISOLATION AND CHARACTERIZATION OF THIOL PROTEINS DURING SPERM CELL MATURATION. Cabrillana ME, Monclus MA, Vincenti AE , Fornés MW, Yuñez R.. IHEM, FCMédicas. UNCuyo - CONICET. Mendoza. mecabrillana@fcm.uncu.edu.ar Sperm thiol oxidation is involved in sperm motility, capacitation, acrosome reaction and fertilization. Changes in thiol oxidation, to disulfide bonds, in sperm cell proteins are verified during epididymal trip. Using different methology we attempt to characterize the thiol reach protein. First, by sonication and ultracentrifugation in sucrose gradient, we fractionated and isolated spermatozoom head and tail. Then compared mature vs. immature sperm cell to establish which specific proteins support SH-SS changes. Second different protocols to solubilize cell proteins were used. Some samples were incubated with detergents (0.1% Triton X100, DTT 32 mM and SDS 4%) and soluble vs non-soluble proteins were analyzed. All samples were incubated with monobromobimane (mbb) that allow staining thiol proteins. Other method was used to localize in sperm cell or in the fraction above mention using the mbb but observed under fluorescence microscopy and were also studied by electron microscopy to know the subcellular origin of the proteins. The thiol proteins were separated by SDS-PAGE and determinated the molecular weight. Proteins about 27 kDa display a great fluorescence as well as the principal piece of the sperm. Complementary at TEM this fraction show elements corresponding to sperm tail. Additional effort will be down to further characterize an isolated one by one of the mbb-SH-SS-proteins.