45Ca2+ Uptake by Spermatozoa
Ca2+ uptake by epididymal spermatozoa was measured according to the method of Coronel and Lardy. Before this measurement, the spermatozoa in the modified Tyrode’s solution (1.0 X 107 cells/ml) were preincubated with 1.5 ^M P12 or recombinant P12 at 37°C for 10 or 60 min. Then they were incubated in the presence of 200 ^M CaCl2 labeled with 45Ca2+ (10-40 ^Ci/mg calcium; Amersham, Arlington Heights, IL) at 37°C for 10 min, and the cells were collected by vacuum filtration under 0.16 atm through a cellulose acetate filter. The cells on the filters were washed with six volumes of modified Tyrode’s solution. The filters were dried and transferred into scintillation vials containing 5.0 ml of scintillation counting cocktail (Merck, Darmstadt, Germany). Radioactivity was counted by a liquid scintillation counter (Parker; LKB, Stockholm, Sweden). buy cheap antibiotics
Assay of P12 Binding to Spermatozoa
P12 was labeled with 125I-Na in the presence of Iodo-bead (Pierce, Rockford, IL) according to previously described method. The radiolabeled P12 was used to characterize the P12-binding site on sperm. Epididymal spermatozoa were incubated with 125I-P12 at 25°C. The cells were collected on a Whatman (Clifton, NJ) GF/C glass microfiber filter by rapid filtration at a pressure of 0.5 atm. The filters had been blocked with 1% nonfat skim milk in 50 mM Tris buffer at pH 7.4 for 30 min and washed with ice-cold buffer before they were used. The filter was washed with six changes of 0.2 ml of ice-cold PBS and air-dried on a filter paper. Radioactivity of the filter was counted by a 7-counter. The nonspecific binding of 125I-P12 to cells was measured by addition of a 100-fold excess of nonradioactive ligand. Each assay was performed in triplicate, and specific binding was the difference between the averages of the total binding and the nonspecific binding.