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

CABRILLANA M, MONCLUS M., VINCENTI A., BURGOS M. Y FORNÉS M.

Uspallata, Mendoza

Congreso; XXIII Reunión Anual de la Soc. de Biol. de Cuyo. III Reunión de la Soc. Argentina de Microscopía; 2005

Sociedad de Biología de Cuyo

Location, cuantification and isolation of sperm proteins with high thiols levels  expressing  during epididymal transit. Cabrillana ME,  Monclus MA, Vincenti AE and Fornés MW IHEM – CONICET, FCM-UNCuyo. E mail: mecabrilana@yahoo.com.ar During epididymal transit the sperms support several changes known as sperm maturation. They allow the gamete to achieve capacitation and ability to fertilize the oocyte. Among the main changes that sperm suffer, the proteins oxido-reduction status (thiols are oxidated to disulfide) detach.  Disulfides bonds are responsible of chromatin condensation and sperm tail structure stabilization. In rat the epididymal fluid is crucial in these processes, due to the oxido-reducting properties of this milieu. Based in our previous experiences in protein isolation and cellular fractionation we could separate and localize subcellular structures rich in thiols groups. Sperm cells from  epididymal caput and cauda, were split in head and tail by sonication at 4 bursts at 30s intervals. After sonication, intact sperm are not longer than 1 %. Head and tail were isolated by sucrose gradient (40 / 80 %). The presence of sperm head in the fraction containing sperm tail was 20 %. Instead the presence of sperm tails in the head fraction was 50 %.  Both fractions were treated with monobrobimane, specific fluorescent thiol labels, then, proteins were extracted with sample buffer (Laemmli UK) and analyzed by SDS-PAGE. Some minor differences were observed mainly in proteins from tail fraction. More effort must be done to acquire a precisely isolation method.