Fig. 2. NO mobilises stored Ca²⁺ in sperm. (A) Spermine NONOate causes a slowly developing rise in [Ca²⁺]_i in human sperm. Responses of four separate cells are shown. Red trace shows example of a cell generating [Ca²⁺]_i oscillations. (B) In low-Ca²⁺ medium ([Ca²⁺]5 µM), the response to NONOate was similar, but oscillations were rarely seen. Responses of seven cells are shown. (C) Pseudocolour image series showing NONOate-induced rise in [Ca²⁺]_i in the sperm neck/midpiece. Numbers show minutes since application of 100 µM spermine NONOate. (D) Mean normalised increase in fluorescence 10 minutes after application of 100 µM spermine NONOate to cells bathed in sEBSS (271 cells; three experiments) and low-Ca²⁺ sEBSS (214 cells; three experiments). (E) A rapid decrease in [Ca²⁺]_i followed washout of NONOate, followed by slow recovery. Upon re-introduction of NONOate, many cells generated oscillations in the neck/midpiece region. Responses of five individual cells shown. Lower panel shows pseudocolour images series of a single [Ca²⁺]_i oscillation (numbers show time in seconds).