237 PROTEIN-TYROSINE PHOSPHORYLATION AND CALCIUM UPTAKE IN BOAR SPERM SUBPOPULATIONS AFTER DIFFERENT DISCONTINUOUS PERCOLL GRADIENT CENTRIFUGATIONS

Abstract

Semen is composed of an heterogeneous population of sperm with varying degrees of structural and functional differentiation. To optimize in vitro fertilization (IVF), different selection methods have been used to isolate sperm subpopulations with high fertilizing capacity. The aim of this study was to evaluate protein-tyrosine phosphorylation (P-Tyr) and calcium uptake in boar sperm subpopulations from the same ejaculate. Spermatozoa from fertile and normozoospermic boars were treated by 2-step discontinuous gradient centrifugation consisting of 3 different combinations of isotonic Percoll (45/60, 60/75, and 45/90%). An aliquot of semen was kept without centrifugation as a control group. Immunofluorescence was employed to determine the localization of proteins phosphorylated in tyrosine residues (Tardif et al. 2001 Biol. Reprod. 65, 784–792). In order to measure intracellular free [Ca²⁺], spermatozoa treated as described above were incubated with 2.5 μM Fura-2AM in a noncapacitating medium (Tardif et al. 2003 Biol. Reprod. 68, 207–213) for 45 min at 37°C. Then, spermatozoa were resuspended in TALP medium (Rath et al. 1999. J. Anim. Sci. 77, 3346–3352), incubated (5% CO₂, 38.5°C) for a further 10 min and then analysed in a fluorescence spectrofluorometer during 30 min with excitation wavelength set at 340–380 nm and emission held at 510 nm. The calculation of intracellular free [Ca²⁺] was performed according to the equation of (Grynkiewicz et al. 1985 J. Biol. Chem. 260, 3440–3450). The results showed that Percoll treatment increased the percentage of cells with P-Tyr v. control group (P < 0.01). Among Percoll groups, 45/90% showed the highest pattern of cells with signal in equatorial region, acrosome, and tail. As was observed with P-Tyr results, the intracellular free Ca²⁺ concentration (nM) was different between control group and spermatozoa treated with Percoll gradient (P < 0.01). Among Percoll groups, 45/90% showed the highest levels in calcium uptake and 45/60 showed the lowest levels (299.26; P < 0.01). In conclusion, it could be stated after Percoll centrifugation that sperm subpopulations have different abilities to protein-tyrosine phosphorylation and calcium uptake that could be related to different levels of in vitro fertility.