Fragmin

Low-molecular weight heparin dalteparin was from injectable Fragmin^® (Pfizer; lot Y08663). BMH was obtained from a commercial supplier (Kin Master, Vila Annes, Brazil). The synthetic pentasaccharide Arixtra^® was from GlaxoSmithKline (London, UK). Heparinases I (EC 4.2.2.7), II and III (EC 4.2.2.8) were purchased from Grampian Enzymes (Aberdeen, Scotland, UK), while recombinant human heparanase, covalently linking the 8 and 50 kDa subunits to produce active single-chain heparanase molecules [42 (link)], was kindly provided by Israel Vlodavsky. All other reagents and chemicals were of HPLC grade or higher quality.

For the decondensation of sperm chromatin we adopted a protocol published elsewhere [36 (link)]. Briefly, aliquots from sorted populations containing approximately 1 x 10⁶ spermatozoa were washed with 4 ml of PBS 1x/0.5% HSA and chromatin stabilization was performed by incubation in 500 μl of 0.5% PFA at 4°C for 10 minutes. Samples were then centrifuged and resuspended in the decondensation solution (PBS 1x containing 5 mM dithiothreitol (DTT, Carl Roth, Karlsruhe, Germany); 0.1% Triton and 100U/ml of low molecular weight heparin (Fragmin, Pfizer, New York, NY, USA) or in PBS containing 0.5% HSA in case of the non decondensed samples. During decondensation (30 minutes in the dark at 25°C), every 10 minutes, spermatozoa were counted under the microscope to exclude loss of cells. Microscopic analysis revealed that the number and the integrity of the PI-dimmer and PI-brighter spermatozoa were maintained during the process. Finally the samples were centrifuged and fixed in PFA 3.7% overnight at 4°C. To verify whether the sperm DNA was efficiently decondensed, aliquots of decondensed and non-decondensed samples were re-stained with PI and flow cytometry analysis was performed. A significant increase in the PI-fluorescence of the decondensed population was observed in all cases. These samples were subsequently used to test the influence of chromatin decondensation in TUNEL results.

SEC experiments were carried out on a Varian ProStar 215 solvent delivery system, coupled to a Varian ProStar 320 UV/VIS detector set at a wavelength of 280 nm. Separation was performed using a BioSep 5-μm SEC-s2000 145 Å, 300 × 4.6-mm LC column (Phenomenex, Torrance, CA). 50 mm sodium phosphate buffer, pH 6.65, containing 150 mm NaCl was used as eluent buffer. Flow rate was set at 0.5 ml/min. Proteins were injected at 0.1 mg/ml concentration in the presence or absence of 1 mg/ml low-molecular-mass heparin (commercial name Fragmin®, Pfizer). Molecular masses of proteins were calculated using the calibration curve provided by the manufacturer.