Thiol reduction and decondensation of sperm nuclei in vivo: cooperative effect of heparin and GSH?

JULIANELLI V, ROMANATO M, CALVO L, CALVO JC

Buenos Aires, IByME

Jornada; XI Jornadas Multidisciplinarias de la Sociedad Argentina de Biología; 2009

SAB

Sperm chromatin decondensation requires a thiol reducing agent and a protamine acceptor. The aim of this study was to analyze the interplay between both agents during thiol reduction and decondensation of human sperm nuclei in vitro. Semen specimens were obtained from normozoospermic (WHO criteria) volunteers. Isolated sperm nuclei were resuspended in HTF-BSA medium and incubated with heparin (Hep) + GSH or DTT at 37°C, adding both reagents either simultaneously (30’) or sequentially: thiol reducer (15’) + wash + heparin (15’) and viceversa. The % decondensed spermatozoa (%Des) was determined by phase contrast microscopy and thiol reduced status of chromatin was evaluated with Acridine Orange. %Des was significantly lower when Hep and GSH were used sequentially, regardless of which reagent was added first. DesR (%Des relative to %Des obtained with Hep + thiol reducer 30’) 27±13% (Hep first) and 40±19% (GSH first) (n=3, ANOVA + Tukey, p<0.05). %Des was not affected by sequential use of reactants when DTT was used as thiol reducer: DesR 82±13% (Hep first) and 97±7% (DTT first) (NS). These results were mimicked by % nuclei with native (low thiol content, green) or denatured (high thiol content, yellow to red) chromatin. We propose the existence of a cooperative effect between protamine acceptor and thiol reducing agent in vivo, where GSH is known to function as thiol reducing agent.