HEPARAN SULFATE AS PUTATIVE DECONDENSING AGENT OF HUMAN SPERMATOZOA IN VIVO: PRELIMINARY EVIDENCE OF ITS PRESENCE IN THE MAMMALIAN OOCYTE.

ROMANATO M, REGUEIRA E, ZAPPI M, DE DIOS C, CALVO L, CALVO JC

Buenos Aires, Argentina

Jornada; VIII Jornadas Multidisciplinarias de la Sociedad Argentina de Biología; 2006

Human sperm decondense in vitro in the presence of heparin (Hep) and glutathione (GSH). Previous studies from our laboratory have shown that heparin sulfate (HS), but not other glycosaminoglycans (GAGs), has a decondensing ability similar to that of Hep in vitro and we have proposed HS as a protamine acceptor in vivo.  The aim of this study was to investigate the presence of HS in the oocyte, via two different strategies. Semen specimens were provided by normospermic (OMS) donors. Oocytes were obtained from the oviduct of 8 weeks –old CF1 female mice, hormonally stimulated with PMSG (5 UI) and hCG (5 UI), and stripped of both cumulus cells (hyaluronidase 0,1% in PBS) and zona pellucida (acid tyrodes, pH=2,5). 1) Methanol fixed oocytes were stained with Rubipy (Tris (2,2´-bipyridine) Ruthenium (II) Chloride) 1 mg/ml in distilled water at pH 1,5 for preferential binding to sulfate groups. 2) Sperm were decondensed on microscope slides with Hep 46 M + GSH 10 mM or mechanically crushed oocytes + GSH 10 mM, for 3.5 h. 1) Analysis by confocal microscopy suggested the presence of sulfated GAGs in the cytoplasm of all oocytes observed. 2) There were no significant differences in sperm decondensation in the presence of oocytes/GSH (38±8%) vs. Hep/GSH (41±6%) (n=7, Paired Student, p=0.25). Addition of heparinase to decondensation milieu significantly inhibited sperm decondensation (3±1% vs. 50±14%) (n=3, Paired Student, p<=0.05) whereas addition of either chondroitinase ABC or hyaluronidase did not (48±11% and 41±8% respectively). These results constitute the first evidence in the literature on the presence of HS in the ooplasm and support its role as protamine acceptor in vivo.