A) Human phospho-kinase array analysis revealed phosphorylation events induced by NAGly (3 μM) after 7 min stimulation. ( B) Shown are the relative phosphorylation (mean pixel intensities relative to control) of two independently performed experiments using two different donor samples. Each array was performed using pooled protein lysates of at least three different semen samples from the same donor. The dotted line indicates the control level. ( C) Western Blot experiments validated the phosphorylation of Akt at the T308 phosphorylation site. Determination of the total amount of Akt served as a control. Furthermore, the quantification of the mean pixel intensity of phosphorylated Akt relative to total Akt upon NAGly stimulation (black bar) is shown. The ratio was normalized to that observed in DMSO-treated cells (0.1%; control; gray bar). Error bar represents the mean ± SEM (p = 0.016, Student’s t-test, n = 4 experiments). ( D) In single-cell calcium imaging experiments, the repetitive short-term stimulation with 10 μM NAGly did not induce any calcium signals in human spermatozoa. Cytosolic Ca 2+ levels are monitored as the integrated f340/f380 fluorescence ratio expressed as a function of time. Black bars indicate the stimulus duration. The positive control (500 nM progesterone) induced strong calcium signals in virtually all sperms and showed the vitality of the sperm and the functionality of the imaging system. In none of the 453 analyzed cells from 4 different sperm samples NAGly induced a Ca 2+ signal.