Wood CD et al. (2003), Speract induces calcium oscillations in the spe...

ECB-ART-38051

J Cell Biol 2003 Apr 14;1611:89-101. doi: 10.1083/jcb.200212053.

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Speract induces calcium oscillations in the sperm tail.

Wood CD , Darszon A , Whitaker M .

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Sea urchin sperm motility is modulated by sperm-activating peptides. One such peptide, speract, induces changes in intracellular free calcium concentration ([Ca2+]i). High resolution imaging of single sperm reveals that speract-induced changes in [Ca2+]i have a complex spatiotemporal structure. [Ca2+]i increases arise in the tail as periodic oscillations; [Ca2+]i increases in the sperm head lag those in the tail and appear to result from the summation of the tail signal transduction events. The period depends on speract concentration. Infrequent spontaneous [Ca2+]i transients were also seen in the tail of unstimulated sperm, again with the head lagging the tail. Speract-induced fluctuations were sensitive to membrane potential and calcium channel blockers, and were potentiated by niflumic acid, an anion channel blocker. 3-isobutyl-1-methylxanthine, which potentiates the cGMP/cAMP-signaling pathways, abolished the [Ca2+]i fluctuations in the tail, leading to a very delayed and sustained [Ca2+]i increase in the head. These data point to a model in which a messenger generated periodically in the tail diffuses to the head. Sperm are highly polarized cells. Our results indicate that a clear understanding of the link between [Ca2+]i and sperm motility will only be gained by analysis of [Ca2+]i signals at the level of the single sperm.

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Genes referenced: LOC100887844 LOC115918576 LOC576642 LOC586260

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	Figure 1. Typical response of a field of sperm to addition of speract to a final concentration of 125 nM. (A) Fluo-4 fluorescence immediately before speract addition. (B) Fluo-4 fluorescence 4 s after speract addition. Images extracted from a time series acquired at 40 frames per second with 25-ms individual frame exposure time. (C) Ratio increases in Fluo-4 fluorescence across whole field. Upper trace (black), response to addition of 125 nM speract at t = 0. Lower trace (red), fluorescence from control field with no speract addition. Both traces have been corrected for the effects of fluorophore bleaching. Data acquired at 40 frames per second with 25-ms individual frame exposure time.
	Figure 2. Ratio increases in [Ca2 + ]i from head and flagellum regions (measured in discrete regions of interest as indicated). Images acquired at 40 frames per second with 25-ms individual frame exposure time. (A) Typical spontaneous fluctuations in [Ca2+]i observed in resting sperm. Images above graphs are intensity images; images below graphs are ratio images against the frame immediately before the spontaneous increase occurred. (B) Typical response of an individual sperm to addition of speract to a final concentration of 125 nM. Images above graphs are intensity images; images below are ratio images against the frame immediately before speract addition. Note the markedly higher intensity of the head compared with the tail and that the fold increases in head and tail are comparable.
	Figure 3. Subregional analysis of [Ca2 + ]i increases in individual sperm. (A) After addition of 100 nM speract, the time taken for the initial calcium increase to reach half height (relative to the first point of increase in the flagellum) was recorded for subregions of the sperm head and flagellum (B); n = 5, error bars indicate ± SEM. One-tailed t test (comparison to flagellum). *, P < 0.001; , P < 0.01; ns, P > 0.05.
	Figure 4. The magnitude of the tonic calcium increase and the frequency of the phasic calcium fluctuations are dependent on the concentration of speract. (A) For each sperm head, the ratio increase in [Ca2+]i was determined from the maximum fluorescence intensity post-speract addition (Fmax) and the initial fluorescence intensity value (F0). Fmax/F0 is presented as the average of all sperm heads analyzed (total number of sperm heads at each speract concentration shown in brackets). Error bars indicate ± SEM. At concentrations of speract above and including 100 pM, only those sperm heads that showed a response to speract were analyzed. At concentrations of speract below and including 10 pM, and in control analyses, sperm heads were chosen at random. (B) Changes in [Ca2+]i were determined for individual flagella by ratioing their fluorescence (F) against their initial fluorescence (F0). The number of fluctuations occurring after speract addition was determined by including all excursions whose increase from trough to peak was >20% of the trough value. Data are presented as the average number of fluctuations occurring in 10 s post-speract addition per flagellum. Number of flagella analyzed indicated in brackets. Error bars represent ± SEM.
	Figure 5. Speract-induced increases in [Ca2 + ]i are dependent on [Ca2 + ]e in a dose-dependent manner. (A) For each sperm head, the ratio increase in [Ca2+]i was determined from the maximum fluorescence intensity after addition of speract to a final concentration of 1 nM (Fmax) and the initial fluorescence intensity value (F0). Fmax/F0 is presented as the average of all sperm heads analyzed (total number of sperm heads at each [Ca2+]e shown in brackets). Error bars represent ± SEM. (B) Typical responses in individual sperm heads at different [Ca2+]e. F/F0 shown over time after addition of speract to a final concentration of 1 nM (indicated by arrow) and data represented as a five-point rolling average. Images acquired at 10 frames per second with 100-ms individual frame exposure time. One-tailed t test (comparison to prior [Ca2+]e) *, P < 0.01; , P < 0.05.
	Figure 6. The degree and characteristics of speract-induced increases in [Ca2 + ]i are altered by both increasing and decreasing [K + ]e. (A) For each sperm head, the ratio increase in [Ca2+]i was determined from the maximum fluorescence intensity after addition of speract to a final concentration of 500 pM (Fmax) and the initial fluorescence intensity value (F0). Fmax/F0 is presented as the average of all sperm heads analyzed (total number of sperm heads at each [K+]e shown in brackets). Error bars represent ± SEM. (B) Typical responses in individual sperm heads at different [K+]e. F/F0 shown over time after addition of speract to a final concentration of 500 pM (indicated by arrow). Images acquired at 10 frames per second with 100-ms individual frame exposure time. One-tailed t test (comparison to 10 mM data) **, P < 0.001; , P < 0.05.
	Figure 7. Representative examples of the effect of Ni2 + treatment on speract-induced [Ca2 +] i increases in individual sperm heads. Blue traces from sperm treated with 300 μM Ni2+ before addition of 500 pM speract (indicated by arrow); red trace from untreated sperm. Changes in [Ca2+]i were determined for individual heads by ratioing their fluorescence (F) against their initial fluorescence (F0). Images acquired at 10 frames per second with 100-ms individual frame exposure time.
	Figure 8. Representative example of the effect of niflumic acid treatment on speract-induced [Ca2 + ]i increases in individual sperm. Sperm treated with 100 μM niflumic acid before addition of 1 nM speract (indicated by arrow). Changes in [Ca2+]i were determined for heads and flagella by ratioing their fluorescence (F) against their initial fluorescence (F0).
	Figure 9. Representative examples of the effect of IBMX treatment on speract-induced [Ca2 + ]i increases in individual sperm. (A) Effect of IBMX treatment on Ca2+ increase in sperm heads. Images acquired at 10 frames per second with 100-ms individual frame exposure time. (B) Effect of IBMX treatment on [Ca2+]i increase in sperm flagella. Images acquired at 40 frames per second with 25-ms individual frame exposure time. In both A and B, blue traces from sperm treated with 100 μM IBMX before addition of 500 pM speract (indicated by arrow); red trace from non-IBMX–treated sperm. Changes in [Ca2+]i were determined for heads and flagella by pseudo-ratioing their fluorescence (F) against their initial fluorescence (F0).
	Figure 10. Quantitative modeling of calcium changes in the head of individual sperm undergoing speract-induced and spontaneous increases in [Ca2 + ]i. For details of the model, see Materials and methods. (A) Typical increase in [Ca2+]i of sperm treated with 100 nM speract (indicated by arrow) with increases in both head and tail. The increase in the head can be modeled by diffusion from the tail through a bottleneck with an apparent rate constant of 2 s−1. (B) Typical increase in [Ca2+]i of sperm undergoing spontaneous fluctuation, with the increase in the head modeled using the same rate constant. Modeling suggests that extrusion or destruction of the diffusing signal is not significant on these timescales. (C) Schematic diagram showing proposed model of speract-induced Ca2+ entry into the heads of sea urchin sperm. Activation of the speract receptor localized to the flagellum results in up-regulation of the activity of adenylate cyclase, also predominantly localized to the flagellum, via increases in pH, [Ca2+]i and/or changes in Em. cAMP then diffuses into the head, opening the cAMP-dependent Ca2+ channels localized there. Calcium efflux pathways are also shown.

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