Article |
Address correspondence to Michael Whitaker, School of Cell and Molecular Biosciences, Medical School, Framlington Place, University of Newcastle upon Tyne, NE2 4HH, UK. Tel.: 44-191-222-5264. Fax: 44-191-222-5164. E-mail: michael.whitaker{at}ncl.ac.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: motility; fluorescence imaging; cyclic nucleotides; cell polarity; membrane potential
A. Darszon is on sabbatical from the Institute of Biotechnology, Department of Genetics and Molecular Physiology, National Autonomous University of Mexico, Cuernavaca, Morelos 62210, Mexico.
* Abbreviations used in this paper: AR, acrosome reaction; [Ca2+]e, extracellular Ca2+; [Ca2+]i, intracellular free calcium concentration; Em, membrane potential; IBMX, 3-isobutyl-1-methylxanthine; [K+]e, external concentration of K+; VDCC, voltage-dependent cation channel.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We undertook high resolution imaging of single sperm to determine whether the measurements of [Ca2+]i in sperm suspensions reflected changes in the sperm head, the sperm tail, or a combination of the two. We found that the earlier measurements by ourselves and others largely reflected [Ca2+]i changes in the head (Schackmann and Chock, 1986; Babcock et al., 1992; Kaupp et al., 2003). Our spatially resolved measurements also revealed previously undiscovered calcium fluctuations in the sperm tail, both spontaneous and speract-dependent. Experiments with ion channel blockers demonstrate that the speract-induced fluctuations are regulated by mechanisms originally uncovered from experiments on sperm suspensions. Thus, speract-induced [Ca2+]i fluctuations are the single cell response that underlies the characteristics of the bulk [Ca2+]i responses measured in sperm suspensions. The spontaneous [Ca2+]i fluctuations, interestingly, do not share this pharmacology and are thus a second new and independent phenomenon. Given these observations, further insights into regulation of [Ca2+]i-modulated sperm motility are likely to be achieved primarily at the level of single sperm.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Sperm with low resting [Ca2+]i may be further divided into speract-responsive and speract-nonresponsive subpopulations, the former comprising between 40100% (median 95%, n = 18) of the sperm with low levels of resting [Ca2+]i. We noted that the proportion of speract nonresponsive cells in each batch of sperm increased as the sea urchin season progressed, though we do not know whether speract responsiveness correlated with the ability of sperm to successfully fertilize eggs.
Fig. 1 B illustrates the same sperm field as in Fig. 1 A, 4 s after exposure to 125 nM speract. Of the cells in Fig. 1 A that have low resting [Ca2+]i, 95% respond to speract addition with an increase in [Ca2+]i. Fig. 1 C shows the Fluo-4 fluorescence increase, as measured across the whole field, in response to addition of 100 nM speract. The peak calcium increase occurs
5 s after speract addition, and persists for upwards of 40 s. This pattern is consistent with previously published observations (Cook et al., 1994; Nishigaki et al., 2001) and indicates that the speract response of adherent sperm is similar to that seen in populations of freely swimming cells. Calcium increases in individual sperm heads were quantified using Fura-2loaded sperm. Resting levels of [Ca2+]i were 364 ± 36 nM, increasing to 1,176 ± 112 nM on addition of 100 nM speract (± SEM, n = 21). These values are consistent with the range of values reported by previous studies (Schackmann and Chock, 1986; Cook and Babcock, 1993b; González-Martinez et al., 2001).
Imaging of single cells reveals spontaneous calcium fluctuations in resting sperm
Around 3% of sperm in the observed field in each experiment undergo spontaneous fluctuations at any given moment, and in such sperm between 1 and 5 fluctuations were recorded over a 10-s period (median = 2, n = 33). As noted above, 95% of sperm with low levels of resting [Ca2+]i respond to speract; their response takes the form of fluctuations in the tail, with a sustained response in the head. Fig. 2 shows the kinetic characteristics of the spontaneous (A) and the speract-induced (B) [Ca2+]i fluctuations. Spontaneous fluctuations have a fast rise time (t1/2 = 100 ± 20 ms) and a relatively slow decay rate (t1/2 = 900 ± 125 ms), and the magnitude and kinetics of these fluctuations are different from those induced by speract (compare Fig. 2 A with Fig. 2 B). Table I is a comparison of the t1/2 of the increase and decay of individual speract-induced and spontaneous fluctuations, and a comparison of their magnitudes. All three criteria are significantly different (P < 0.001; unpaired two-tailed t test), suggesting that the mechanisms involved in the two types of [Ca2+]i fluctuation are distinct (though certain ionic transporters could be shared).
|
|
|
We have not observed any obvious correlation between sperm undergoing spontaneous fluctuations and those that respond to speract; sperm that have displayed spontaneous fluctuations can respond to speract, and cells that have not shown spontaneous fluctuations before speract addition may subsequently respond.
Analysis of calcium fluctuations in different areas of sperm indicates that the initial calcium increase propagates from flagella to head
Fig. 3 illustrates how the speract-induced [Ca2+]i elevation appears to initiate in the flagella and spread from there to the head. The t1/2 of the initial [Ca2+]i elevation increases progressively with distance from the flagella (Fig. 3 A), and there is an 1-s interval between the attainment of peak [Ca2+]i in the flagellum and the tip of the head (Fig. 3 B; n = 5). Subsequent fluctuations show similar kinetic characteristics, as do spontaneous calcium fluctuations (unpublished data). These findings, together with those in Fig. 2, suggest that the [Ca2+]i increase in the head results from the summation of [Ca2+]i and/or cyclic nucleotide changes that occur in the flagella.
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Calcium increases occur first in the sperm tail, then in the head
Close inspection of both spontaneous and speract-induced calcium increases showed that an interval of 1 s elapsed between the calcium increase in the tail and that at the tip of the head; the increase in the flagella was synchronous at the 25-ms time resolution of our experiments. Analysis of the calcium increase in response to speract in head and tail revealed that the response comprised two elements: a tonic response on which was superimposed phasic fluctuations. In the tail, the phasic component predominated, whereas in the head, the tonic component was much more marked, with phasic fluctuations superimposed on a monotonically increasing component that rose to a plateau. As speract concentrations were increased, the phasic component showed an increased frequency (measured in the tail), whereas the tonic component showed an increased magnitude (measured in the head), both over the range of speract concentrations known to modulate the calcium response from experiments in sperm suspensions.
It occurred to us that a simple explanation that might account for these results was that a signal originated periodically in the tail in response to speract and diffused to the head, where it was summed or integrated. This simple hypothesis is supported by quantitative modeling (Fig. 10), though by itself this by no means constitutes proof.
|
Ionic basis of the tonic and phasic responses
Both tonic and phasic components of the calcium response to speract were reduced proportionately by reduction of extracellular calcium, disappearing at 0.5 mM [Ca2+]e, consistent with previous work in suspensions (Schackmann and Chock, 1986). This result demonstrates that the phasic responses in the tail and the tonic response in the head both require calcium influx. High potassium sea water abolished the speract response completely, as has been reported previously (Lee and Garbers, 1986; Schackmann and Chock, 1986; Babcock et al., 1992); under these conditions, the only intracellular response to speract is an increase in cGMP (Harumi et al., 1992).
The calcium channel blocker Ni2+ abolishes the phasic response to speract, but not the tonic response. The anion channel blocker niflumic acid markedly enhances the phasic responses in both head and tail and reduces their frequency.
These observations suggest a mechanism in which the phasic tail responses are sensitive to membrane potential and ion fluxes. A simple hypothesis consistent with these finding is that the fluctuating calcium responses in the tail are driven or terminated by membrane potential changes, and that a component of this mechanism is driven by calcium influx through a Ni2+-sensitive voltage-gated calcium channel in the tail, but essentially absent in the head because the tonic response in the head is unaffected by Ni2+. The enhancement of the fluctuations after treatment with niflumic acid suggests that the membrane potential changes can be modulated by inhibition of anion channels.
Role of cyclic nucleotides
It is generally accepted that the first response to speract in sperm is the generation of cGMP, through activation by the speract receptor of a guanylate cyclase localized to the tail. Changes in pHi and Em then activate an adenylate cyclase (Beltrán et al., 1996), mainly found in the flagella (Toowicharanont and Shapiro, 1988), that has been postulated to open cAMP-activated Ca2+ channels (Cook and Babcock, 1993b). We treated sperm with IBMX, a phosphodiesterase inhibitor that in sea urchin sperm prevents the hydrolysis of cGMP and cAMP (Cook and Babcock, 1993a). We found that the phasic response was abolished completely in the tail and replaced by a small, sustained Ca2+ increase, whereas in the head Ca2+ rose around six times more slowly than in controls, but to levels comparable or greater than the control maximum. These results suggest that the cAMP-gated Ca2+ influx mechanism is more prominent in the head than in the tail. It is interesting to note that in A. punctulata sperm suspensions, the kinetics of the Ca2+ response are much more rapid after uncaging caged cGMP than after uncaging caged cAMP (Kaupp et al., 2003).
A model for the spatial regulation of the Ca2+ response in single sperm
Our analysis of Ca2+ signaling patterns in response to speract in single sperm suggest four main hypotheses. The first is that Em changes in the tail are linked to the predominantly tail-localized Ca2+ fluctuations. The second is that the voltage-dependent calcium channels that drive the fluctuations are located in the tail, as the tail fluctuations can be blocked by inhibitors of certain voltage-activated Ca2+ channels (Ni2+, and to a lesser degree nimodipine), whereas the tonic increases in the head, though dependent on Ca2+ influx, are unaffected. The third is that the activation of the calcium pathway invariably spreads from tail to head, suggesting that the early components of the response that drive the fluctuations are restricted to the tail. The fourth is that preventing the hydrolysis of cGMP and cAMP lead to loss of tail fluctuations and a much slower tonic rise in calcium in the head.
These hypotheses suggest a model in which the cGMP-induced membrane potential changes generate tail calcium fluctuations and drive the production of cAMP, which then diffuses from the tail into the head. The model is supported by the observation that in high K+ sea water, which allows only the initial cGMP increase but prevents changes in both membrane potential and cAMP (see model in Cook and Babcock, 1993b), speract addition fails to generate a calcium increase in the tail, let alone the head.
The importance of the fluctuations in driving the head calcium response is underlined by the consequences of treatment with IBMX. Though levels of both cGMP and cAMP are enhanced (Cook and Babcock, 1993b; Beltrán et al., 1996), the calcium response (and so presumably the cAMP response) is very substantially slowed.
It is interesting to consider the mechanism by which the head might integrate the speract response. One obvious possibility is that calcium increases in the tail diffuse slowly into the head region. This seems to us unlikely on two grounds. The first is that the calcium concentrations in head and tail seem comparable, not differing by the 10-fold or more that would be necessary to fit a calcium diffusion model. The second is that we have detected no gradient of calcium concentration in the tail. Because the tail is long and thin and has a much smaller volume than the head, it is difficult to see how substantial diffusion of calcium from tail to head could occur without the appearance of a gradient in the tail. A more attractive explanation is that the diffusing vector is cAMP, generated as pulses in the tail in synchrony with the calcium fluctuations. cAMP then gates cAMP-dependent calcium channels preferentially located in the head. Why does the head integrate the tail-derived signals? Our model suggests that it is because there is a diffusion bottleneck at the point that the tail joins the head. Other explanations are possible: for example, the calcium extrusion mechanisms may have a lower capacity in the head than in the tail, or phosphodiesterase activity may be lower in the head than the tail. Because calcium decreases faster during oscillations in the tail than in the head, we favor the former as more reasonable; the surface/volume ratio of the head is far higher than the tail, so that the condition would apply even at constant extrusion activity per membrane surface area. In fact, it is possible that intrinsic extrusion rates are higher in the tail than the head, for although the sperm Ca2+-ATPase (Garcia-Soto et al., 1988) localization is unknown, it has been shown that a potassium-dependent Na+/Ca2+ exchanger (suNCKXT) is localized to the tail and mitochondrial region and is absent in the head (Su and Vacquier, 2002). Moreover, modulating Na+/Ca2+ exchanger activity alters the kinetics of the speract response in cell populations (Rodriguez and Darszon, 2003). The latter explanation is also possible, as our simple model shows that there little loss of the diffusing substance in the head itself; decreases in the head are modeled by diffusion back into the tail through the bottleneck, rather than by degradation in the head. A model for the spatial localization of the elements of the speract response mechanism is shown as Fig. 10 C.
Speract and motility
It remains to be shown that speract acts in sperm-egg chemotaxis like its analogue resact (Ward et al., 1985; Kaupp et al., 2003). Nonetheless, there is evidence that speract can modulate sperm motility (Cook et al., 1994). Modulation of sperm swimming behavior is thought to occur at the level of the axoneme, and indeed, calcium is known to modulate the beat characteristics of sea urchin sperm (Brokaw, 1987; Cosson, 1996). The generally unspoken assumption in experiments in which calcium is measured in sperm populations is that speract-induced calcium increases are occurring in the tail, in contrast to those involved in the acrosome reaction that take place in the head (Cook et al., 1994; Darszon et al., 2001). It is now clear that the calcium signal measured in experiments with sperm populations is predominantly from the head. It seems very likely that the phasic tail responses that we have uncovered will play a vital role in modulating sperm swimming behavior in response to speract.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials
Artificial seawater (ASW) contained (mM): 430 NaCl, 10 KCl, 10 CaCl2, 23 MgCl2, 25 MgSO4, 2 NaHCO3, and 1 EDTA (pH 8.0, 9501000 mosM). Low Ca2+ ASW (pH 7.0) was as ASW but at pH 7.0 and with 1 mM CaCl2, and Ca2+-free ASW was ASW with no added CaCl2. K+-free ASW was prepared as ASW but omitting KCl, and high K+ ASW was prepared as ASW but with 20 mM KCl. Fura-2 AM, Fluo-4 AM, and pluronic F-127 were from Molecular Probes, Inc. IBMX, ionomycin, NiCl2, niflumic acid, and poly-D-lysine were from Sigma-Aldrich. Speract was synthesized in Dr. Possani's laboratory as described previously (Nishigaki et al., 2001).
Loading of fluorescent indicators (AM esters) into sperm
Dry sperm were suspended in 10 volumes of low Ca2+ ASW (pH 7.0) containing 0.5% wt/vol pluronic F-127 and 20 µM of either Fura-2 AM or Fluo-4 AM and incubated for 23 h at 16°C. Loaded sperm were then diluted with 10 volumes low Ca2+ ASW (pH 7.0), centrifuged for 10 min at 1,000 g and 4°C, and resuspended in the original volume of low Ca2+ ASW (pH 7.0). Sperm were stored in the dark and on ice until use.
Single-cell imaging
Imaging chambers were prepared by coating coverslips with 50 µg/ml poly-D-lysine, shaking off excess, and allowing to air-dry. Coated coverslips were then attached to Perspex rings with high vacuum silicon grease to form watertight chambers. Labeled sperm were diluted 1:20 to 1:40 in ASW, immediately placed into the chambers, and left for 13 min, after which unattached sperm were removed by washing with ASW. The chambers were then filled with 1 ml of the appropriate medium (if not ASW, 3 x 1-ml washes were performed with appropriate medium). Chambers were then mounted on a microscope (Diaphot 300; Nikon) with a 40x fluor objective, and fluorescence changes were recorded on a camera (Coolsnap-FX; Photometrics) used in continuous (stream) acquisition mode.
Image processing
Images were processed offline using MetaMorph® (Universal Imaging). Rolling background subtraction was performed using a region of interest placed as close as possible to the sperm of interest. Any incompletely adhered sperm that moved during the course of any experiment were discounted. Fluorescence measurements in individual sperm were made by manually drawing a region of interest around the head or a section of flagella for each sperm. Measurements were taken from head or flagella regions as indicated in the figure legends. All measurements of speract-induced or spontaneous fluctuations were taken from flagella. A fluctuation was defined as an excursion whose increase from trough to peak was >20% of the trough value.
Determination of [Ca2+]i
The majority of data presented were obtained using Fluo-4loaded sperm. These are presented as pseudoratios as indicated on the individual figures. [Ca2+]i was determined from Fura-2 measurements according to the following formula: [Ca2+]i = Kd(R - Rmin)/(Rmax R)(Fb380/Ff380). The ratiometric 340/380 value Rmax and Fb380 (fluorescence at 380 excitation) were obtained when calcium-saturated by the addition of 2 µM ionomycin in ASW. Rmin and Ff380 were obtained after addition of 2 µM ionomycin in Ca2+-free ASW supplemented with 10 mM EGTA. Kd = 774 nM (Poenie et al., 1985).
Modeling of [Ca2+]i increases in sperm heads
It was assumed that calcium changes in the head were driven by diffusion of a substance down a concentration gradient from tail to head and that a second process removed the substance from the head by extrusion or metabolism. An approximate model of these processes is provided by the differential equation: dy/dt = k1 · (u - y) k2 · y, where y is the concentration of a substance in the head and u is the concentration of the same substance in the tail. k1 and k2 are rate constants. This differential equation was solved using a Runge-Kutta approach.
![]() |
Acknowledgments |
---|
The work was supported by grants from the Wellcome Trust to M.J. Whitaker and A. Darszon, and by grants from Consejo Nacional de Ciencia y Tecnologia and DGAPA/UNAM to A. Darszon.
Submitted: 6 December 2002
Revised: 13 February 2003
Accepted: 24 February 2003
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arnoult, C., J.R. Lemos, and H.M. Florman. 1997. Voltage-dependent modulation of T-type calcium channels by protein tyrosine phosphorylation. EMBO J. 16:15931599.
Babcock, D.F., M.M. Bosma, D.E. Battaglia, and A. Darszon. 1992. Early persistent activation of sperm K+ channels by the egg peptide speract. Proc. Natl. Acad. Sci. USA. 89:60016005.[Abstract]
Beltrán, C., O. Zapata, and A. Darszon. 1996. Membrane potential regulates sea urchin sperm adenylylcyclase. Biochemistry. 35:75917598.[CrossRef][Medline]
Brokaw, C.J. 1979. Calcium-induced asymmetrical beating of triton-demembranated sea urchin sperm flagella. J. Cell Biol. 82:401411.[Abstract]
Brokaw, C.J. 1987. Regulation of sperm flagellar motility by calcium and cAMP-dependent phosphorylation. J. Cell Biochem. 35:175184.[Medline]
Cardullo, R.A., S.B. Herrick, M.J. Peterson, and L.J. Dangott. 1994. Speract receptors are localized on sea urchin sperm flagella using a fluorescent peptide analog. Dev. Biol. 162:600607.[CrossRef][Medline]
Cook, S.P., and D.F. Babcock. 1993a. Selective modulation by cGMP of the K+ channel activated by speract. J. Biol.Chem. 268:2240222407.
Cook, S.P., and D.F. Babcock. 1993b. Activation of Ca2+ permeability by cAMP is coordinated through the pHi increase induced by speract. J. Biol. Chem. 268:2240822413.
Cook, S.P., C.J. Brokaw, C.H. Muller, and D.F. Babcock. 1994. Sperm chemotaxis: egg peptides control cytosolic calcium to regulate flagellar responses. Dev. Biol. 165:1019.[CrossRef][Medline]
Cosson, J. 1996. A moving image of flagella: news and views on the mechanisms involved in axonemal beating. Cell Biol. Int. 20:8394.[CrossRef][Medline]
Darszon, A., C. Beltran, R. Felix, T. Nishigaki, and C.L. Trevino. 2001. Ion transport in sperm signaling. Dev. Biol. 240:114.[CrossRef][Medline]
Garcia-Soto, J., M. Mourelle, I. Vargas, L. de De la Torre, E. Ramirez, A.M. Lopez-Colome, and A. Darszon. 1988. Sea urchin sperm head plasma membranes: characteristics and egg jelly induced Ca2+ and Na+ uptake. Biochim. Biophys. Acta. 944:112.[Medline]
Gauss, R., R. Seifert, and U.B. Kaupp. 1998. Molecular identification of a hyperpolarization-activated channel in sea urchin sperm. Nature. 393:583587.[CrossRef][Medline]
González-Martinez, M.T., A. Guerrero, E. Morales, L. de De La Torre, and A. Darszon. 1992. A depolarization can trigger Ca2+ uptake and the acrosome reaction when preceded by a hyperpolarization in L. pictus sea urchin sperm. Dev. Biol. 150:193202.[Medline]
González-Martinez, M.T., B.E. Galindo, L. de De La Torre, O. Zapata, E. Rodriguez, H.M. Florman, and A. Darszon. 2001. A sustained increase in intracellular Ca2+ is required for the acrosome reaction in sea urchin sperm. Dev. Biol. 236:220229.[CrossRef][Medline]
Harumi, T., K. Hoshino, and N. Suzuki. 1992. Effects of sperm-activating peptide I on Hemicentrotus pulcherrimus spermatozoa in high potassium sea water. Dev. Growth Differ. 34:163172.
Kaupp, U.B., J. Solzin, E. Hildebrand, J.E. Brown, A. Helbig, V. Hagen, M. Beyermann, F. Pampaloni, and I. Weyand. 2003. The signal flow and motor response controling chemotaxis of sea urchin sperm. Nat. Cell Biol. 5:109117.[CrossRef][Medline]
Lee, H.C., and D.L. Garbers. 1986. Modulation of the voltage-sensitive Na+/H+ exchange in sea urchin spermatozoa through membrane potential changes induced by the egg peptide speract. J. Biol. Chem. 261:1602616032.
Morales, E., L. de la Torre, G.W. Moy, V.D. Vacquier, and A. Darszon. 1993. Anion channels in the sea urchin sperm plasma membrane. Mol. Reprod. Dev. 36:174182.[Medline]
Nishigaki, T., F.Z. Zamudio, L.D. Possani, and A. Darszon. 2001. Time-resolved sperm responses to an egg peptide measured by stopped-flow fluorometry. Biochem. Biophys. Res. Commun. 284:531535.[CrossRef][Medline]
Poenie, M., J. Alderton, R.Y. Tsien, and R.A. Steinhardt. 1985. Changes of free calcium levels with stages of the cell division cycle. Nature. 315:147149.[Medline]
Rodriguez, E., and A. Darszon. 2003. Intracellular sodium changes during the speract response and the acrosome reaction in sea urchin sperm. J. Physiol. 546:89100.
Schackmann, R.W., and P.B. Chock. 1986. Alteration of intracellular [Ca2+] in sea urchin sperm by the egg peptide speract. Evidence that increased intracellular Ca2+ is coupled to Na+ entry and increased intracellular pH. J. Biol. Chem. 261:87198728.
Su, Y.H., and V.D. Vacquier. 2002. A flagellar K(+)-dependent Na(+)/Ca(2+) exchanger keeps Ca(2+) low in sea urchin spermatozoa. Proc. Natl. Acad. Sci. USA. 99:67436748.
Suarez, S.S., S.M. Varosi, and X. Dai. 1993. Intracellular calcium increases with hyperactivation in intact, moving hamster sperm and oscillates with the flagellar beat cycle. Proc. Natl. Acad. Sci. USA. 90:46604664.[Abstract]
Toowicharanont, P., and B.M. Shapiro. 1988. Regional differentiation of the sea urchin sperm plasma membrane. J. Biol. Chem. 263:68776883.
Ward, G.E., C.J. Brokaw, D.L. Garbers, and V.D. Vacquier. 1985. Chemotaxis of Arbacia punctulata spermatozoa to resact, a peptide from the egg jelly layer. J. Cell Biol. 101:23242329.[Abstract]