Capacitation of hamster spermatozoa with the divalent cation chelators D-penicillamine, L-histidine, and L-cysteine in a protein-free culture medium.

By using a chemically defined (protein-free) culture medium that supports sperm viability but not capacitation or the acrosome reaction, we have determined that hamster spermatozoa can be chemically capacitated in vitro by the divalent cation chelators D-penicillamine, L-histidine, and L-cysteine in the absence of bovine serum albumin (BSA). Washed cauda epididymal spermatozoa were preincubated (1-2 x 10(6) sperm/ml) for 3, 4, or 6 hr at 37 degrees C in 5% CO2 in air. The basic culture medium used for sperm preincubation and for sperm:egg coincubation was a modified Tyrode's solution (protein-free) containing 10 mM sodium lactate, 100 microM sodium pyruvate, and 1.0 mg/ml polyvinylalcohol (TLP-PVA). Sperm viability was maintained in all preincubation and coincubation media with PHE (20 microM D-penicillamine, 100 microM hypotaurine, and 1.0 microM epinephrine). The low control sperm preincubation medium consisted of TLP-PVA. In some cases the high control preincubation medium also contained 3 mg/ml BSA (TALP-PVA). The experimental preincubation medium was TLP-PVA with additional D-penicillamine (125 or 500 microM), or L-histidine (10, 100, or 1,000 microM) or L-cysteine (25, 75, or 125 microM). After preincubation, sperm were coincubated (2 x 10(4) sperm/ml) with cumulus-free hamster eggs in TALP-PVA +/- additional D-penicillamine, L-histidine, or L-cysteine for 1.5 hr, fixed, and evaluated for percent egg penetration as an index of sperm capacitation. The results demonstrate that hamster spermatozoa can be chemically capacitated in vitro with D-penicillamine (500 microM: range of mean penetration values, 53.6%-84.3%), L-histidine (100 microM: range of mean values, 24.8%-56.3%) or L-cysteine (75 microM: 51.3%) in the absence of exogenous protein.