Location, isolation and characterization of protein with thiol groups detected during maturation in sperm

CABRILLANA ME;; MONCLUS MA; VINCENTI AE; FORNES MW

Potrero de los Funes, San Luis

Congreso; XXVI Reunión Científica Anual de la Sociedad de Biología de Cuyo.; 2006

Sociedad de Biología de Cuyo.

Oocyte fertilization involves mature and capacitated sperm. The sperm support maturation during the epididymal transit and capacitation during the stadium in the female genital tract. Changes in thiol oxidation to disulfides bonds, in sperm proteins are verified during epididymal trip. Instead, the capacitation status is assumed by an increment in tyrosine phosphorylated sperm proteins (p-Y). Using sonication and ultracentrifugation in sucrose gradient we fractionated and isolated sperm heads and tails from mature vs. immature sperm cells. Then incubation of fractions with fluorescent monobromo bimane (MMB) permitted thiols proteins stain. The cellular location of these proteins was established in sperm head or tail by microscopy and molecular weight standard SDS-PAGE. Parallel gel was also run to perform an immune blot for p-Y. These gels were used to compare the thiol or/and p-Y amount. Some samples from the sucrose gradient were incubated with 1% Triton X100 and soluble vs. non soluble proteins were analyzed with the protocol above mentioned. Phosphorylated and MBB marked proteins were coincident in mature and capacitated sperm and were isolated in soluble fraction of sperm. Results indicate that same proteins are stabilized during maturation by thiol changes and phosphorylation during capacitation