LOCATION, QUANTIFICATION AND ISOLATION OF HIGH THIOL LEVEL SPERM PROTEINS EXPRESSING DURING EPIDIDYMAL TRANSIT

CABRILLANA ME; MONCLUS MA; VINCENTI AE; FORNÉS MW

Uspallata, Mendoza

Congreso; Reunión Conjunta de: XXIII Reunión Científica Anual de la Sociedad de Biología de Cuyo y III Reunión Científica de la Sociedad Argentina de Microscopía (SAMIC); 2005

Sociedad de Biología de Cuyo

During epididymal transit, the sperms undergo several changes, collectively known as sperm maturation. It allows the gamete to achieve capacitation and ability to fertilize the oocyte. One major chance affects sperm protein redox status, namely that thiol groups are oxidized to form disulfide bonds. Disulfide bonds are responsible of chromatin condensation and sperm tail structure stabilization. In rats, the epididymal fluid is crucial in this process, due to the redox properties of this milieu. Based in our previous experiences in protein isolation and cellular fractionation, we isolated and localized subcellular structures rich in thiol groups. Sperm cells from epididymal caput and cauda were split in head and tail by 4 bursts of sonication at 30-s intervals. After sonication, no more than 1% of sperm remain intact. Heads and tails were isolated by sucrose gradient (40 – 80%). The presence of sperm head in fraction containing sperm tail was 20%, while the presence of sperm tail in the head fraction was 50%. Both fractions were treated with monobromobimane, a specific fluorescent thiol label. Afterwards, proteins were extracted with sample buffer (Laemmli UK) and analyzed by SDS-PAGE. Some minor differences were observed, mainly in proteins from tail fraction. Further work must be done to improve the isolation method