The epididymal spermatozoa were flushed from the cauda of bovine epididymides with a perfusion of cold PBS through the deferent duct. Spermatozoa were washed twice in PBS, recovered by centrifugation (600-700 rpm, 4 min Beckman gs-6R centrifuge; Beckman), and resuspended in fresh PBS. Typically, most spermatozoa were motile. For immunolabeling they were diluted 1:5 in PBS. For immu-noblot analyses, spermatozoa were diluted 1:1 in cold PBS containing 1 mM PMSF and sonicated while on ice using a VWR Sonifier II Cell Disrupter Branson Ultrasonics at maximal setting during three consecutive intervals of 30 sec each. proventil inhaler
Thanks to the kind help of Dr. Yves Brindle, freshly ejaculated bovine spermatozoa were obtained from the Centre d’Insemination Artificielle du Quebec (CIAQ; Sainte Madeleine, PQ, Canada). A total of 10 ejaculates were collected from fertile bulls by means of an artificial vagina. The ejaculates were not pooled; each ejaculate was assessed individually for volume, appearance, and motility. Samples showing less than 70% motile cells were discarded. Samples were diluted 1:1 in a modified Tyrode’s medium (TALP) described by Bavister and Yanagimachi (100 mM NaCl, 3.1 mM KCl, 25 mM NaHCO3, 0.29 mM KH2PO4, 21.6 mM lactic acid, 11.5 mM MgCl2, 1 mM pyruvate, 10 mM HEPES, pH 7.4). They were washed twice in TALP medium. Cells were recovered by centrifugation (600-700 rpm, 4 min Beckman gs-6R centrifuge).