Fig. 8. Intracellular manganese clearance in human spermatozoa. One of three repeat experiments in which Fura-2-loaded human spermatozoa were suspended in sEBBSS containing 1 mM MnCl₂ and stimulated with progesterone. Measurement of fluorescence at 360 nm (isobestic point for Fura-2) shows rapid quenching of fluorescence due to the initial rapid progesterone-induced Mn²⁺ influx followed by a slower quench due to the subsequent slower influx. Subsequent addition of 1 mM La³⁺ (to block Mn²⁺ influx) not only prevented further quench but resulted in partial recovery of fluorescence. The initial fast phase (see inset showing detail of response upon La³⁺ application) is probably, at least in part, artefactual (see text). A subsequent slower phase appears to reflect removal of Mn²⁺ from the cytoplasm, consistent with activity of a Mn²⁺ transporting pump (such as SPCA1) in spermatozoa. Subsequent application of 40 µM bis-phenol caused a rapid, partial reversal of the fluorescence recovery with an amplitude corresponding to the slow phase.