142 INTRACELLULAR CALCIUM UPTAKE AS A RESPONSE TO DIFFERENT CAPACITATION TREATMENTS IN EJACULATE AND EPIDIDYMAL BOAR SPERMATOZOA

Abstract

Both cauda epididymal and ejaculated spermatozoa are considered fully mature, although these two types of spermatozoa do not necessarily show the same behavior in vitro. It has been reported that both types of spermatozoa fertilize eggs in vitro at the same rate, but, in general, epididymal ones achieve this objective easier than the ejaculated ones. Intracellular Ca²⁺ influx is one of the crucial biochemical events occuring capacitation and Ca²⁺ requirements for capacitation varies among different species. In this study, we investigated how different source of spermatozoa (ejaculated vs. epididymal) and commonly employed sperm capacitating methods can affect calcium uptake. Sperm-rich fractions from seven fertile boars and sperm from seven different epididymides were used. Semen samples were kept unwashed (method A), washed in Dulbecco's phosphate-buffered saline (DPBS) supplemented with 0.1% BSA (method B), or washed on a Percoll gradient (method C) (Matás et al. 2003 Reproduction 125, 133-141). To measure intracellular free Ca²⁺, spermatozoa, treated as described above, were incubated with 2.5 ¼m fura-1/AM in a non capaciting medium (Tardif et al. 2003 Biol. Reprod. 68, 207-213) for 45 min at 37°C. Then, spermatozoa were resuspended in TALP medium, incubated (5% CO₂, 38.5°C) for a further 60 min and then analyzed in a fluorescence spectrofluorometer with excitation wavelength set at 340-880 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). Results showed that calcium uptake is affected by treatment and semen source (P < 0.001). The intracellular free Ca²⁺ concentrations (nM) in ejaculated semen and in epididymal spermatozoa were 269.52 vs. 208.52, 1134.58 vs. 137.37 and 1224.79 vs. 216.54 for A, B, and C methods, respectively. As a conclusion, it can be stated that sperm capacitation treatment affects calcium uptake. In addition, capacitation pathways may be modified or regulated in some way by seminal plasma, thus increasing intracellular calcium levels in ejaculated sperm (methods B and C) in comparison to those in epididymal spermatozoa.

This work was supported by Ministerio de Educación y Ciencia, AGL2003-03144.