CONICET | Buscador de Institutos y Recursos Humanos

Treatment of sperm with heparin and reduced glutathione (Hep-GSH) induces nuclear sperm decondensation in vitro. GSH is an endogenous disulfide bond reducer which plays a critical role in sperm decondensation and male pronuclear formation in mammals, but its action needs to be followed by the remotion of protamines from DNA. In amphibians and Drosophila melanogaster, nucleoplasmin has been shown to exert this role. In human, heparan sulfate (HS) has been demonstrated to function as protamine acceptor during sperm decondensation in vitro, suggesting that decondensation mechanisms are not completely conserved across the evolutionary scale. Heparin induces the release of histone 1 and protamines (critical step for decondensation), and it is widely used in bovine to induce sperm capacitation before or during in vitro fertilization. Moreover, Hep-GSH treatment has been successfully applied for ICSI by our group and other laboratories. Given that heparin is not present in bovine oocytes, and there is a high similarity between heparin and HS molecules, we hypothesize that HS could act as a protamine acceptor during bovine sperm decondensation in vivo, as has already been postulated by others both in human and mice. To this aim, we studied the decondensation kinetics of semen samples from 6 different bulls after treatment with Hep-GSH, and tested the presence of HS in bovine oocytes. Methods: Frozen sperm from six bulls were thawed individually, and incubated in BO medium containing 80 µM Hep and 15 mM GSH for 1, 3, 7 and 20 h under standard IVM conditions. For each bull, a control group incubated in BO medium was also included. After incubation, treated sperms were fixated with glutaraldehyde and observed under bright light at 400X magnification. The percentage of decondensed spermatozoa was determined, considering as decondensed those sperm with head size > 7 µm and altered membranes. A minimum of 200 cells from each sample were evaluated in duplicate. To investigate the presence of HS in the bovine oocyte, cumulus oocyte complexes were collected from slaughtered cow ovaries and matured in vitro. After 22 h, cumulus cells and zona pellucidae were removed from oocytes at metaphase II (MII) stage. After fixation, indirect immunofluorescence was performed. Briefly, MII oocytes were permeabilized with 0,2% Triton X-100 for 15 min at room temperature. Following incubation in blocking solution for 30 min, oocytes were immunolabeled with anti-HS monoclonal antibody overnight at 4°C. A FITC-goat polyclonal anti-mouse IgM was used as second antibody for 90 min at 37°C. Negative controls did not contain first antibody. Nuclei were counterstained with propidium iodide. Oocytes were mounted on glass slides and observed under confocal microscopy. Data was statistically analyzed by Fisher?s exact test (p