Characterization of Flagellar Cysteine-Rich Sperm

CABRILLANA ME; MONCLUS MA; SAEZ LANCELOTTI TE; BOARELLI PV; CLEMENTI MA; VINCENTI AE; YUNES RM; FORNES MW

CELL MOTILITY AND THE CYTOSKELETON

WILEY-LISS, DIV JOHN WILEY & SONS INC

Año: 2011 p. 1 - 10

0886-1544

Mammalian sperm proteins undergo thiol groups (SH) oxidation to form disulfides bonds (S-S) as they travel through the epididymis during cell maturation. Disulfide bonds are involved in chromatin condensation and tail organelles stabilization. In this work, we used a fluorescent thiol-selective labeling agent, monobromobimane (mBBr), to study the protein thiol status of rat sperm during maturation. Fluorescence signal decrease along the epididymal trip, more evidently in head but also in tail, indicating that both sub cellular regions participate in the thiol changes. The sources of fluorescence signal are sulfhydrils sperm proteins labeled by mBBr (mBBr-spp). Initial attempt to identify mBBr-spp labeled were detected in the initial ?caput- but not in the distal ?cauda- segment of the epididymis in SDS-PAGE analysis. This phenomenon could be due to protein resistance to solubilization. For this reason disulfides bonds reduction was accomplished by sodium dodecyl sulphate plus dithiothreitol treatment to recover the mBBr signal in SDS-PAGE. Under this protocol, a major 27 kDa protein band, display a strong signal. Protein identification by mass spectrometry and sequence database searching correlated this protein with the outer dense fiber 1 (ODF1). mBBr specifically bound to N-terminal domanin cysteine of ODF1. mBBr reduce rat sperm motility, quantitatively and qualitatively, and the effects are dose dependent, without significal increasing the percentage of dead sperm. Thus, we found that ODF1 is highly responsible for mBBr fluorescence detection in sperm tail and the motility inhibition by the fluorescence marker indicate that ODF1 N-terminal domain are related to sperm motility.