The objective of this study was to determine if human ejaculated sperm exhibit caspase 3 activation in response to physiological stimuli. Human semen was obtained from healthy volunteers at the Extremadura Center of Human Assisted Reproduction of Badajoz with local ethical committee approval. Samples were collected after 4-5 days of abstinence, and were allowed to liquefy for 30 min. To determine caspase-3 activity, stimulated or resting cells were lysated and incubated with caspase-3 substrate (AC-DEVD-AMC), and measured with a fluorescence spectrophotometer with excitation wavelength of 360 nm and emission at 460 nm.

The treatment of spermatozoa with 20 mM progesterone for 30 min induced a significant increase in caspase-3 activity (133.5±22.7 % above control, p<0.05, n=10). The effect of progesterone on caspase activation was time dependent, reaching a maximal caspase activity after 60 min of stimulation at 20 mM with a increase of 144.5±31.7 % above control (n=4). Loading of cells with 10 mM dimethyl BAPTA, an intracellular Ca²⁺ chelator, for 30 min significantly reduced progesterone-induced caspase activation (144.5±31.7% versus 111.3±4.5 % above control, p<0.05). Similar results were obtained with 10 mM Ru360, a specific blocker of calcium uptake into mitochondria. Cell treatment for 90 min with 100 mM z-LEHD-FMK (an inhibitor of caspase-9) abolished progesterone-induced caspase-3 activation.

We conclude that progesterone induces caspase-3 activation in human spermatozoa through calcium signal dependent mechanisms.

This work was supported by Laboratorios SERONO, S.A.