Biol. Bull. 205: 185-186. (October 2003)
© 2003 Marine Biological Laboratory
Ryanodine-Sensitive Calcium Flux Regulates Motility of Arbacia punctulata Sperm
D. E. Heck1,* and
J. D. Laskin2
1 Rutgers University, Piscataway, NJ
2 UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ
* Corresponding author: heck{at}eohsi.rutgers.edu
The motility of sea urchin spermatozoa and the direction of their movement are due to the beat of the flagellum, which is controlled by an ion flux across the plasma membrane. When sea urchin sperm contact egg jelly, channels are activated that rapidly (<5 s) and transiently elevate intracellular calcium, initiating a cascade of events that lead to the sperm acrosome reaction (1–3). This process, in turn, induces a complex sequence of events that include protein phosphorylation and elevation of calcium levels thought to be dependent on intracellular calcium mobilization (4). Recent reports have identified ryanodine-gated intracellular calcium stores as critical for controlling the motility of mammalian sperm (5). In the present studies, we determined whether increases in intracellular calcium and motility in the sperm of the sea urchin Arbacia punctulata that are mediated by egg-derived products are also mediated by ryanodine-gated intracellular calcium stores.
In initial studies, we found that Arbacia punctulata egg-derived products (egg water) that stimulate sperm motility also increase sperm intracellular free calcium (Fig. 1, panel A). The increase was about 10-fold, occurred within 2 min, and persisted for at least 10 min. These experiments and all further experiments were repeated three times with similar results. The increase in intracellular calcium did not require calcium in the seawater, indicating that the ion was likely released from internal stores (data not shown). In further experiments, therefore, we used calcium-free seawater. Sperm motility and increases in intracellular free calcium were also found to be stimulated 2- to 3-fold by ryanodine (0.3–1 µM) or caffeine (1–3 µM), both agonists of ryanodine-gated ion channels (for reviews see refs. 5 and 6) (data not shown). We next examined the effects on sea urchin sperm of ruthenium red, a potent inhibitor of ryanodine-sensitive channel activity (6–8). We found that 1 µM ruthenium red would inhibit the calcium mobilization and sperm motility initiated by egg water, or by ryanodine and caffeine (Fig. 1, panel C and not shown). Taken together, these data indicate that a ryanodine-sensitive calcium flux regulates the motility of Arbacia punctulata sperm.
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Figure 1. Effects of egg water and SIN-1 on calcium mobilization in sperm from the sea urchin Arbacia punctulata. Freshly isolated sea urchin sperm were incubated with the cell permeant calcium-sensitive fluorescent indicator fluo-4 AM (Molecular Probes, Eugene, OR, 5 µM, 10 min) and then stimulated with egg water (panels A and C) or SIN-1 (Molecular Probes, 1 µM, panels B and D). After 3 min, intracellular levels of free calcium were analyzed using a Coulter EPICS flow cytometer (excitation wavelength 488 nm, emission wavelength 525 nm). Data are presented on a 4-decade log scale. US, unstimulated sperm. In experiments where cells were treated with ruthenium red, the sperm were first transiently permeabilized using a Live Cell Permeabilization Kit (Gibco, Grand Island, NY) to allow uptake of the compound (panels C and D). Note that sperm treated with ruthenium red failed to mobilize calcium in response to egg water or SIN-1 (panels C and D). (Lower panel): Schematic representation of events leading to activation of Arbacia punctulata sperm. Events originate with the binding of egg-derived mediators to their receptors on the surface of the sperm and progress through signaling to stimulate the release of calcium from internal stores. This release potentially involves protein interaction with intracellularly produced nitric oxide and presumably occurs within the endoplasmic reticulum, which mediates the opening of high-capacity sodium channels in the plasmalemma and ultimately leads to motility. PM, plasma membrane; RYR, ryanodine receptor; ER, endoplasmic reticulum; NO, nitric oxide.
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Recent studies indicate that ryanodine receptors are regulated by nitric oxide-induced oxidation and reduction of critical sulfhydryl residues (9, 10). In previous studies, we and others have demonstrated that nitric oxide is also important in regulating motility in sea urchin sperm, and we have found further that this process is likely to be dependent on the activity of GTP-binding proteins (11–13). In these reports, sperm motility and nitric oxide production were inhibited by cholera toxin, and motility was recovered by the addition of the nitric oxide/peroxynitrite-releasing drug 3-morpholinosydnonimine (SIN-1). We found that SIN-1 (1 µM) also stimulated sperm calcium mobilization (Fig. 1, panel B). Increases in intracellular calcium were approximately 3- to 4-fold, persisted for at least 10 min, and were also independent of extracellular calcium. Motility and calcium mobilization induced by SIN-1 in Arbacia punctulata sperm were also found to be inhibited by ruthenium red (Fig. 1, panel D, and not shown). These data indicate that nitric oxide, like egg water, is effective at mobilizing intracellular calcium. Moreover, nitric oxide appears to function in sea urchin sperm upstream of ryanodine-sensitive calcium channels.
The lower panel in Figure 1 shows a schematic representation of events leading to the activation of Arbacia punctulata sperm. In this model, the interaction of the egg-derived mediator with sperm receptors activates the guanylate cyclase activity of the receptor, resulting in the formation of cGMP. The resulting cascade of events, including an intermediate calcium-induced release of sequestered intracellular calcium ("gray box") that provokes the activity of high capacity sodium channels in the plasmalemma, ultimately culminates in motility. We hypothesize that nitric oxide, produced in response to enzymatic binding of calcium/calmodulin made available by the initial brief rise in intracellular calcium, interacts with ryanodine-gated ion channels to mediate the release of calcium from internal stores. We speculate that, in a manner similar to that of ryanodine-gated release of calcium from stores in mammalian skeletal muscles (7, 14), nitric oxide-mediated alterations to channel proteins are central to the prolonged and hyperactive motility of egg mediator-activated sperm.
Literature Cited
- Ward, G. E., D. L. Garbers, and V. D. Vacquier. 1985. Science 227: 768–770.[Abstract/Free Full Text]
- Schackmann, R. W., and P. B. Chock. 1986. J. Biol. Chem. 261: 8719–8728.[Abstract/Free Full Text]
- Su, Y. H., and V. D. Vacquier. 2002. Proc. Natl. Acad. Sci. USA 99: 6743–6748.[Abstract/Free Full Text]
- Schackmann, R. 1986. Methods Cell Biol. 27: 57–71.[ISI][Medline]
- Walensky, L. D., T. M. Dawson, J. P. Steiner, D. M. Sabatini, J. D. Suarez, G. R. Klinefelter, and S. H. Snyder. 1998. Mol. Med. 4: 502–514.[ISI][Medline]
- Ehrlich, B. E., E. Kaftan, S. Bezprozvannaya, and I. Bezprozvanny. 1994. Trends Pharmacol. Sci. 15: 145–149.[Medline]
- Salama, G., E. V. Menshikova, and J. J. Abramson. 2000. Antioxid. Redox Signal. 2: 5–16.[Medline]
- Ozawa, T. 2001. Int. J. Mol. Med. 7: 21–25.[ISI][Medline]
- Galione, A., and G. C. Churchill. 2002. Cell Calcium 32: 343–354.[ISI][Medline]
- Heck, D. E. 2001. Antioxid. Redox Signal. 3: 249–260.[ISI][Medline]
- Heck, D. E., W. Troll, and J. D. Laskin. 1996. Biol. Bull. 191: 275–276.[ISI]
- Heck, D. E., W. Troll, and J. D. Laskin. 1996. Pp. 16–17 in The Biology of Nitric Oxide V, S. Moncada, S. Gross, J. Stamler, and A. Higgs, eds. Portland Press, Portland, ME.
- Herrero, M. B., E. de Lamirande, and C. Gagnon. 2003. Curr. Pharm. Des. 9: 419–425.[ISI][Medline]
- Sun, J., L. Xu, J. P. Eu, J. S. Stamler, and G. Meissner. 2003. J. Biol. Chem. 278: 8184–8189.[Abstract/Free Full Text]
