Figure 2.

Flexstation assay development. Capacitated spermatozoa were pre-loaded with calcium-sensitive fluorescent dyes and progesterone evoked responses measured. Progesterone evoked fluorescence increased with increase in sperm cell concentration (A). Calcium 3 dye had a larger assay window compared with Fluo3-AM due to higher amplitude of fluorescence (B). Calcium 3 dye had a larger assay window compared with Calcium 5 dye, due to lower background fluorescence (C). Amplitude of the progesterone evoked response was inversely proportional to the extracellular calcium concentration (D). Progesterone elicited responses were unaffected by DMSO at concentrations up to 3% (v/v) (E). Progesterone evokes a concentration-dependent increase in intracellular calcium in human sperm cells (F).