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Fig. 1. Single cell imaging of testosterone-induced intracellular Ca²⁺ oscillations. (A) Fluorescence sequence over the time period indicated (images in pseudocolor). The Ca²⁺ signal began at the extreme tip of the cell and propagated as a Ca²⁺ wave. A few seconds later a new Ca²⁺ wave was produced, generating Ca²⁺ oscillations. (B) Normalized Ca²⁺ changes induced by 10 nM testosterone. (C) Cells stimulated with 100 nM testosterone. The time of addition of the hormone is indicated (arrows in B and C). (D) Power spectral analysis shows that the dominant peak of testosterone-induced Ca²⁺ oscillations occurs at 22 mHz. (E) Concentration dependence of the amplitude of the testosterone-induced Ca²⁺ transient, with a maximum response produced at 100 nM testosterone. (F) 17ß-estradiol (red line) but not 17-estradiol (green line) induces Ca²⁺ transients. Cell pre-incubated with 1 mM tamoxifen, an antagonist of estrogen receptor, did not modify the testosterone-induced Ca²⁺ oscillations, indicating that the estrogen pathway is not required and the response was specific for the testosterone effect. (G) The linescan mode of recording fluorescence intensity was used to monitor different cellular regions. The x axis shows time, read from left to right. The y axis is the distance within the cell shown on the far left. The dashed lines, labeled 1 to 3, are the representative regions plotted in panel H. (H) Transient rises in Ca²⁺ were observed at different times, showing a cytosolic Ca²⁺ wave that propagated towards the nucleus, into the nucleus, and often to the opposite end of the cell. ^*P<0.05.

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