Measurement of calcium influx in surface-fixed single sperm cells: efficiency of different immobilization methods.

Abstract

Calcium fluxes across the plasma membrane of spermatozoa are part of the signal transduction pathway during sperm capacitation. To identify the topography, the time sequence and frequency of calcium fluxes in motile human spermatozoa, individual spermatozoa have to be locally immobilized in a measurement chamber to allow the quantification of ionized calcium by use of a microspectro-photometric method (FURA-2AM). In this study, we compared different immobilization methods using agarose-, gelatin-, laminin- and poly-L-lysine-coated glass slides. Optimal results were obtained with poly-L-lysine coating, which permitted the adhesion of motile spermatozoa that could be analysed microspectro-photometrically. The loss of adherent spermatozoa during the washing procedures was below 10%. However, a major disadvantage of poly-L-lysine coating is that this polymer itself induces a low calcium flux in spermatozoa. Comparing all tested variations, laminin offered the best adhesion result without any detectable effects on calcium fluxes. Our method allowed the rapid change of incubation fluid and calcium concentrations around individual motile spermatozoa and the reproducible quantification of calcium fluxes in single motile spermatozoa.