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Abstract

  1. Top of page
  2. Abstract
  3. CHEMOATTRACTANTS AND THEIR RECEPTORS
  4. CELLULAR SIGNALING PATHWAY
  5. MOTOR RESPONSE AND NAVIGATION STRATEGY
  6. OUTLOOK
  7. Acknowledgements
  8. LITERATURE CITED

Sperm are attracted by chemical substances which are released by the egg. This process is called chemotaxis. Several molecules that are involved in chemotactic signaling of sperm from marine invertebrates are described and a model of the signaling pathway is presented. We discuss the motor response during chemotaxis and propose a model of the navigation strategy of sperm. J. Cell. Physiol. 208: 487–494, 2006. © 2006 Wiley-Liss, Inc.

Sperm adjust their swimming path in a gradient of a chemical factor released by the egg—a process called chemotaxis. The gradient of the chemoattractant provides cues that guide sperm to the egg. Most of our knowledge on sperm chemotaxis originates from the study of marine invertebrates, mainly sea urchin and starfish. In particular the genera Arbacia, Strongylocentrotus, and Asterias have advanced to model systems for the study of chemotactic signaling. Comparative studies indicate that the signaling pathway and motor response are highly conserved among echinodermata (Matsumoto et al., 2003; Solzin et al., 2004; Böhmer et al., 2005). Therefore, this review is restricted to sperm chemotaxis of species from this phylum. Several molecules have been proposed to be involved in chemotactic signaling—among them cyclic AMP, cyclic GMP, Ca2+ ions, and protons (for reviews see Garbers, 1989b; Darszon et al., 1999; Darszon et al., 2001; Neill and Vacquier, 2004). However, their physiological function and the sequence of cellular events are still debated. In mammals, the receptors and intracellular messengers are not known for certain. The interested reader is referred to a comprehensive review by Eisenbach and Giojalas (2006).

It is well established that binding of chemoattractants to receptors on the sperm surface gives rise to changes in the concentration of intracellular Ca2+ ([Ca2+]i) (for review see Darszon et al., 2005). Ca2+ influx is a crucial step; in the absence of external Ca2+, chemotaxis is abolished (Cosson et al., 1984; Miller, 1985; Ward et al., 1985; Kaupp et al., 2003). The Ca2+ dynamics in the flagellum controls the swimming trajectory of sperm (Böhmer et al., 2005; Wood et al., 2005). Elaborate models have been proposed to account for the bewildering complexity of cellular reactions that seem to be involved in the regulation of [Ca2+]i (Darszon et al., 1999, 2001, 2005). Notably, cyclic nucleotides and intracellular pH (pHi) have been proposed to control Ca2+ entry.

Kinetic techniques such as “stopped-flow,” “quenched-flow,” and flash photolysis of caged compounds (cyclic nucleotides as well as chemoattractants), in combination with Ca2+ imaging have recently been employed to test these models (Nishigaki et al., 2001; Kaupp et al., 2003; Wood et al., 2003; Solzin et al., 2004; Böhmer et al., 2005; Wood et al., 2005). The use of caged chemoattractants allowed for the first time to study the motor response and the navigation strategy of sperm in a defined chemical gradient (Böhmer et al., 2005). Finally, the genome project of the sea urchin Strongylocentrotus purpuratus provides an invaluable source for the identification of molecules involved in the chemotactic response (Su and Vacquier, 2002; Galindo et al., 2005; Nomura et al., 2005; Su and Vacquier, 2005).

The most important questions are: what is the nature of chemoattractants and their respective receptors? Which signaling pathway controls the Ca2+ dynamics? How do sperm adjust their swimming trajectory during chemotaxis? Finally, how do sperm navigate in a gradient of the chemoattractant? In the following, we discuss recent advances that are related to these questions.

CHEMOATTRACTANTS AND THEIR RECEPTORS

  1. Top of page
  2. Abstract
  3. CHEMOATTRACTANTS AND THEIR RECEPTORS
  4. CELLULAR SIGNALING PATHWAY
  5. MOTOR RESPONSE AND NAVIGATION STRATEGY
  6. OUTLOOK
  7. Acknowledgements
  8. LITERATURE CITED

About 80 peptides that affect sperm motility have been identified mainly from two different phyla—cnidaria and echinodermata (Miller, 1985; Suzuki, 1995). These peptides are referred to as sperm-activating peptides (SAPs). Only for two SAPs, the chemotactic function has been unequivocally demonstrated: one is resact, a 14 amino-acid peptide from the sea urchin Arbacia punctulata (Ward et al., 1985; Kaupp et al., 2003; Böhmer et al., 2005), the other is asterosap, a 34 amino-acid peptide from the starfish Asterias amurensis (Nishigaki et al., 1996; Böhmer et al., 2005). The receptors for these peptides are guanylyl cyclases (GCs) that synthesize cGMP from GTP after binding of the respective peptide (Ramarao and Garbers, 1985; Shimomura et al., 1986; Nishigaki et al., 2000; Matsumoto et al., 2003).

Although, the function of most SAPs has not been firmly established for some of them, it is tacitly assumed that they are involved in chemotaxis. Ironically, even for speract from S. purpuratus, the most intensely studied SAP, the function is not known. Although speract, like resact, activates a cGMP-signaling pathway, its receptor reportedly is unrelated to GCs (Dangott et al., 1989). Moreover, attempts failed to demonstrate chemotactic activity of speract (Cook et al., 1994; Solzin et al., 2004). Considering that the cGMP-signaling pathways of sperm from Arbacia and Strongylocentrotus are astoundingly similar (Cook and Babcock, 1993a,b; Cook et al., 1994; Kaupp et al., 2003; Matsumoto et al., 2003; Nishigaki et al., 2004; Solzin et al., 2004; Böhmer et al., 2005; Shiba et al., 2005; Wood et al., 2005), it is odd that these species seem to employ different receptors. We propose to re-examine the identity of the speract receptor and to inquire whether this peptide binds to a receptor GC as well. In a similar vein, we propose to re-examine the chemotactic activity of speract or to search for the chemoattractant of Strongylocentrotus sperm.

Are all SAPs chemoattractants and do chemoattractants subserve additional functions? Some peptides like speract, for which no chemotactic activity has been reported, may fail in chemotaxis assays for several reasons. For example, in mammals, chemotactic activity shows a bell-shaped dependence on the concentration of the chemoattractant (see Eisenbach & Giojalas, 2006), because chemotaxis is attenuated when the receptor becomes saturated. The range of physiological concentrations of chemoattractants is not known for certain; therefore, previous studies may have employed concentrations that were too high. Furthermore, the chemotactic response may be transient and some assays may fail to detect rapid and transient responses.

SAPs are derived from larger precursors, and processing by proteases gives rise to several isoforms. For example, a single mRNA encodes several copies of speract and six related isoforms (Ramarao et al., 1990). Isoforms with a C-terminal sequence GGGVG stimulate respiration. Other cellular reactions have not been tested. The function, if any, of these isoforms is not known. Similarly, an N-terminal peptide derived from startrak, a 13 kD protein isolated from ovaries of the starfish Pycnopodia helianthoides, is chemotactically active (Miller and Vogt, 1996) and displays a significant sequence similarity to several isoforms of asterosap isolated from mature oocytes of A. amurensis (Nishigaki et al., 1996). These isoforms enhance sperm motility (Nishigaki et al., 1996), but their chemotactic activity has not been tested.

SAPs seem to affect various other sperm functions including general motility, respiration, and the acrosome reaction (Garbers, 1989a). In previous experiments, high concentrations (up to 100 µM) of peptides have been employed, which exceed the concentration that saturates the peptide-induced Ca+ influx by several orders of magnitude (Kaupp et al., 2003; Matsumoto et al., 2003; Solzin et al., 2004). In retrospect, responses evoked by high SAP concentrations may reflect protective or adaptive reactions (to cope with excessive stimulation) rather than functional diversity. Notwithstanding, at low and high concentrations, peptides indeed may serve distinct functions. For example, asterosap, the chemoattractant of Asterias, controls Ca2+ entry and sperm guidance at low concentrations and facilitates the acrosome reaction at high concentrations (Nishigaki et al., 1996; Matsumoto et al., 2003; Böhmer et al., 2005).

CELLULAR SIGNALING PATHWAY

  1. Top of page
  2. Abstract
  3. CHEMOATTRACTANTS AND THEIR RECEPTORS
  4. CELLULAR SIGNALING PATHWAY
  5. MOTOR RESPONSE AND NAVIGATION STRATEGY
  6. OUTLOOK
  7. Acknowledgements
  8. LITERATURE CITED

Chemoattractants, and also speract, activate multiple cellular reactions; among them are changes in pHi, in the concentrations of cyclic nucleotides, Na+ and Ca2+ ions, in membrane voltage (Vm), and in the phosphorylation pattern of several proteins. Authentic and presumed functions of these reactions have been assembled into sophisticated models of chemotactic signaling (Cook et al., 1994; Darszon et al., 1999, 2001; Shiba et al., 2005). However, the sequence of events and the specific function of several cellular reactions are controversial (Kirkman-Brown et al., 2003; Eisenbach, 2004). In the following, we will discuss some of the most pertinent issues.

The cyclic-nucleotide signal

Chemoattractants evoke a large and rapid increase of the intracellular concentration of cGMP. The cGMP level increases by 30- to 100-fold within 200–400 msec and decays to almost resting values within 10 sec (Kaupp et al., 2003; Matsumoto et al., 2003). The rapid rise of cGMP represents the primary event in chemotactic signaling. Cyclic GMP has been proposed to activate a K+ channel in the flagellar membrane of sea urchin sperm, thereby causing a hyperpolarization of the cell (Lee and Garbers, 1986; Babcock et al., 1992; Cook and Babcock, 1993a; Galindo et al., 2000).

In Arbacia and Strongylocentrotus, the increase of the cAMP concentration is smaller than the increase of cGMP (Kaupp et al., 2003; Matsumoto et al., 2003) and in Asterias it is insignificant (Matsumoto et al., 2003). Moreover, the elevation of cAMP compared to cGMP requires higher concentrations of the chemoattractant and it proceeds slower. Previous models ascribe to cAMP a central role in the control of Ca2+ entry (Cook and Babcock, 1993b; Cook et al., 1994; Darszon et al., 1999, 2001, 2005; Wood et al., 2003). The release of cAMP from its caged compound elicits a Ca2+ influx (Kaupp et al., 2003; Nishigaki et al., 2004), a finding that would support this idea. However, kinetic analysis rules out the possibility that the resact- or speract-induced rise of cAMP controls Ca2+ entry (Kaupp et al., 2003; Matsumoto et al., 2003). First, in sperm of A. punctulata, the cAMP response is delayed with respect to the cGMP response and does not occur earlier than the Ca2+ response (Kaupp et al., 2003). Second, asterosap, the chemoattractant of A. amurensis, does not increase the concentration of cAMP (Matsumoto et al., 2003). Third, the chemoattractant- and the cGMP-induced Ca2+ signals are similar but distinctively different from the Ca2+ signal evoked by cAMP (Kaupp et al., 2003). Thus, cAMP is still a molecule in search of a function in sperm.

Likewise, the identity of the adenylyl cyclase (AC) that becomes active on stimulation by chemoattractants, its location in the cell, and its mechanism of activation are not clear. It has been proposed that the AC is activated by the peptide-induced hyperpolarization of the membrane (Beltrán et al., 1996), that is, that the AC is voltage-sensitive. A flagellar 180 kD calmodulin-binding protein from Strongylocentrotus (Bookbinder et al., 1990) has recently been identified as a member of the family of soluble AC (sAC) (Nomura et al., 2005). In general, sACs from various species are activated by Ca2+ and HCOmath image (Chen et al., 2000; Jaiswal and Conti, 2003; Litvin et al., 2003), and the structural basis of this regulation, which does not involve CaM, has been elucidated (Steegborn et al., 2005). Quite unexpected, the purified sAC from Strongylocentrotus is not Ca2+ sensitive, but becomes active at alkaline pHi (Nomura et al., 2005). This finding would support a previous suggestion that the AC activity is enhanced by the peptide-induced alkalinization (Cook and Babcock, 1993b). However, definitive conclusions as to the function of cAMP and the relative importance of the two forms of ACs—soluble AC versus transmembrane AC—have to await further clarification.

The Ca2+ signal

Fluorescent dyes along with kinetic techniques that allow resolution of early events were used to monitor the influx of Ca2+ (Kaupp et al., 2003; Matsumoto et al., 2003; Wood et al., 2003; Nishigaki et al., 2004; Wood et al., 2005). Stimulation of sperm either by chemoattractants or by intracellular release of cyclic nucleotides from their caged compounds evokes a rapid and transient increase of [Ca2+]i. Sperm are exquisitely sensitive: a Ca2+ signal is detected at concentrations of resact as low as ∼125 fM and the signal saturates at approximately 25 pM. In fact, sperm respond to the binding of a single molecule of chemoattractant (Kaupp et al., 2003).

A remarkable feature of the Ca2+ response is its pronounced delay ranging from 200 to 600 msec (Kaupp et al., 2003). When the Ca2+ response is evoked by a step increase of cGMP, the delay is shortened to about 150 msec. This minimum delay may result from intervening step(s) required for the opening of Ca2+ channels. One of the candidates for such a step is the hyperpolarization by a cGMP-activated K+ channel. At low concentrations of resact, the rate-limiting step may be the synthesis of cGMP. The delay of the Ca2+ response is functionally important as it determines the timing of the motor response (see below).

Another conspicuous feature is the waveform of the Ca2+ signal. At resact concentrations larger than ∼10 pM, the decay of the Ca2+ signal displays a “hump” which develops into a second phase of Ca2+ entry that persists for a few minutes. The two distinct kinetic phases of the Ca2+ influx have been referred to as “early” and “late” signals (Kaupp et al., 2003). When sperm are stimulated by a step increase of cGMP, the Ca2+ signal fades away in up to four distinct peaks or humps (Kaupp et al., 2003 and unpublished). The waveform may manifest a damped Ca2+ oscillation. Stimulation with speract of single immobilized sperm cells from S. purpuratus evokes persistent Ca2+ oscillations (Wood et al., 2003). Furthermore, on stimulation with resact or cGMP, successive Ca2+ spikes and corresponding changes in the trajectory are observed in single sperm cells from A. punctulata (Böhmer et al., 2005); stimulation with cGMP of S. purpuratus, however, produces only a single Ca2+ spike or turn (Wood et al., 2005).

While early work reported an increase of [Ca2+]i on stimulation by chemoattractants (Schackmann and Chock, 1986; Cook and Babcock, 1993b; Cook et al., 1994), behavioral studies suggested an initial decrease of [Ca2+]i rather than an increase. The trouble is: studies using sperm, whose membrane had been rendered permeable by treatment with detergent, revealed that the waveform of flagellar beat depends on the [Ca2+] in the medium. At low [Ca2+], flagella beat more symmetrically than at high [Ca2+] (Brokaw et al., 1974; Brokaw, 1979). Thus, flagella beat asymmetrical when [Ca2+]i is high and hence sperm swim on curved trajectories, whereas the beat becomes symmetrical when [Ca2+]i is low and sperm swim on straight trajectories. Furthermore, sperm swim on more or less straight trajectories towards a source of chemoattractant and turn when swimming away from it (Miller, 1985). From these observations it was inferred that an increase of the chemoattractant lowers [Ca2+]i, while a decrease of chemoattractant elevates [Ca2+]i.

Sophisticated models have been proposed to reconcile the swimming behavior with the Ca2+ measurements that seemingly contradict each other. The models predict an initial decrease of [Ca2+]i due to the export of Ca2+ by voltage-dependent Na+/Ca2+ exchange, followed by an increase of [Ca2+]i due to increases in cAMP and pHi (Cook and Babcock, 1993a,b; Cook et al., 1994; Darszon et al., 1999, 2001; Shiba et al., 2005). In fact, an initial decrease of [Ca2+]i might have been escaped detection by studies that were lacking time resolution. Recently, this issue was revisited using fast kinetic techniques. These attempts yielded mixed results. Nishigaki et al. (2004) recorded a small initial decrease of fluorescence that was interpreted as a drop of [Ca2+]i. The decrease was followed by a 100-fold larger increase of fluorescence (Nishigaki et al., 2004). Occasionally, we also observed a small decrease of fluorescence with both cGMP and cAMP (T. Strünker and U.B. Kaupp unpublished), but not with resact (Kaupp et al., 2003). We are reluctant to interpret this minute and variable change in fluorescence as a Ca2+ signal. Whatever the correct interpretation might be, an initial decrease of [Ca2+]i is not behaviorally relevant, because the swimming trajectory does not change during the first 200 msec after the release of cGMP (see below) (Kaupp et al., 2003; Böhmer et al., 2005; Wood et al., 2005). We propose that the early Ca2+ influx is the crucial cellular event that determines chemotactic behavior.

The ion channel through which Ca2+ flows into the cell has not yet been identified. It is also unclear, whether the early and late phases of the Ca2+ entry are mediated by different ion channels. Because changes in [Ca2+]i evoked by the chemoattractant are attenuated by dihydropyridines, it has been proposed that voltage-activated Ca2+ channels (Cav), in particular high-threshold L-type channels, cause Ca2+ entry (Wood et al., 2003; Nishigaki et al., 2004; Granados-Gonzalez et al., 2005; Wood et al., 2005). The applied concentrations of blockers, however, were 2–3 orders of magnitude higher than those required to inhibit mammalian Cav channels. Complementary DNA fragments amplified from testis of S. purpuratus show significant sequence similarity with voltage-activated Ca2+ channels Cav 1.2 and Cav 2.3 from mammalia (Granados-Gonzalez et al., 2005). The absence of a Ca2+ response at high external [K+] (Granados-Gonzalez et al., 2005; Wood et al., 2005), which depolarizes the membrane, also supports the idea that voltage-activated Ca2+ channels are involved.

The pH signal

Protons are considered key players in the physiology of sperm (for reviews see Shapiro and Tombes, 1985; Garbers, 1989b; Ward and Kopf, 1993; Darszon et al., 1999, 2001). A change in pHi has been proposed to initiate sperm motility (Goldstein, 1979; Christen et al., 1982; Christen et al., 1983; Johnson et al., 1983; Lee et al., 1983) and to regulate Ca2+ influx (Cook and Babcock, 1993a; Cook et al., 1994) and the acrosome reaction (Trimmer et al., 1986; Guerrero and Darszon, 1989; Kawase et al., 2005). Furthermore, several enzymatic reactions have been shown to be pH-sensitive, including dephosphorylation of GC and the synthesis and degradation of cAMP by sAC and a phosphodiesterase (PDE), respectively (Suzuki and Garbers, 1984; Suzuki et al., 1984; Cook and Babcock, 1993a,b; Nomura et al., 2005).

In fact, SAPs stimulate a rapid intracellular alkalinization (Schackmann and Chock, 1986; Babcock et al., 1992; Nishigaki et al., 2001; Solzin et al., 2004). Early work led to the notion that a Na+/H+ exchange mechanism, stimulated by hyperpolarization, mediates H+ efflux from the cell (Lee, 1984a,b; Lee, 1985; Lee and Garbers, 1986). A key observation that seems to support this hypothesis was the inhibition of H+ efflux by high external K+ (Harumi et al., 1992). While these early reports were pioneering the study of pH in sperm physiology, the interpretation of the results is not clear-cut. The experiments were done with osmotically swollen sperm or isolated flagella in Na+-free seawater. The ionic gradients and the membrane voltage are entirely undefined under these conditions and are likely to be significantly different from those of intact cells in normal seawater. Moreover, high K+ concentrations were used to abolish the pHi response. Challenging intact sperm with high K+ in a stopped-flow apparatus produced a depolarization followed by a sizeable Ca2+ entry (A. Helbig and U.B. Kaupp, unpublished).

The Na+/H+ exchange mechanism is not supported by recent studies using intact sperm under physiological conditions. If a cGMP-induced hyperpolarization activates Na+/H+ exchange, a pHi signal should be evoked both by chemoattractants and by cGMP. Contrary to this prediction, a step increase of intracellular cGMP gives no pHi response in Arbacia and only a small response in Strongylocentrotus (Solzin et al., 2004). This result is incisive, as it suggests that the proton sink is located upstream of cGMP. Probably it is consumption of GTP that triggers an increase of pHi rather than synthesis of cGMP per se. Furthermore, at physiological concentrations of resact (below 25 pM), the Ca2+ influx precedes the pHi signal. The finding that the pHi signal follows the Ca2+ signal implies that the increase of pHi can occur under depolarizing conditions, that is, when Ca2+ is entering the cell. Moreover, experimental manipulations that abolish the alkalinization neither affect the Ca2+ response nor chemotaxis (Solzin et al., 2004). In light of these novel results, one may wonder whether the peptide-induced pHi signal represents a side reaction with no specific function rather than an essential step in chemotactic signaling. Biochemical reactions in the cytosol that consume protons or changes in mitochondrial activity could produce the alkalinization.

The voltage signal

The elucidation of sensory signaling has greatly benefited from electrophysiological techniques that allow recording of ionic currents across the cell membrane under well-defined conditions. Unfortunately, due to technical difficulties, changes in membrane voltage and the underlying currents cannot be routinely recorded from intact sperm (for review see Darszon et al., 2005). Instead, fluorescent potentiometric probes have been employed to record electrical activity (Babcock et al., 1992; Beltrán et al., 1996; Galindo et al., 2000). However, these studies suffer from several shortcomings that render their interpretation equivocal. Most importantly, the rapid Ca2+ transients (Nishigaki et al., 2001; Kaupp et al., 2003; Matsumoto et al., 2003; Wood et al., 2003; Nishigaki et al., 2004; Solzin et al., 2004; Böhmer et al., 2005; Wood et al., 2005) are difficult to reconcile with the slow and long-lasting changes in membrane voltage that have been recorded with potentiometric dyes (Babcock et al., 1992; Beltrán et al., 1996; Galindo et al., 2000). These studies used swollen sperm in unphysiological saline. Hence, the intracellular ion concentrations, including [Ca2+]i and the resting voltage were undefined. Furthermore, owing to their mechanism of voltage sensing, these potentiometric dyes exhibit slow response times and therefore, may misrepresent the true voltage response.

The HCN channel

Sea urchin sperm harbor in their flagellar membrane an ion channel that belongs to the family of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels. Two different HCN channels have been localized to the sperm flagellum (Gauss et al., 1998; Galindo et al., 2005). A channel from membranes of S. purpuratus sperm reconstituted in planar bilayers displays some properties that are reminiscent of HCN channels (Labarca et al., 1996). HCN channels become activated at a hyperpolarized state of the membrane (Vm more negative than ∼ −40 mV), and their open probability Po at a given voltage is enhanced by cAMP (for reviews see Pape, 1996; Kaupp and Seifert, 2001; Robinson and Siegelbaum, 2003). Although their relative permeability is 3- to 4-fold larger for K+ than for Na+ ions, under physiological conditions (high Na+ outside, high K+ inside), these channels give rise to a depolarizing inward current carried by Na+ ions.

HCN channels may serve multiple functions in sperm. First, a small fraction of HCN channels is constantly open independent of voltage (Gauss et al., 1998; Proenza et al., 2002). Owing to this property and the unique ion selectivity (reversal voltage Vrev of ca. −25 to −35 mV), HCN channels may co-determine the resting voltage Vrest. Second, HCN channels after the cGMP-induced hyperpolarization may depolarize the cell again, and, thereby, initiate the opening of voltage-activated Cav channels. Third, the HCN channel from sea urchin is set apart from its mammalian cousins by two properties that could be functionally important. Sperm HCN channels first activate and than inactivate after a hyperpolarizing voltage step. Cyclic AMP removes this inactivation (Gauss et al., 1998), and consequently, affects Po much more profoundly than in mammalian HCN channels; those stay open on hyperpolarization and cAMP shifts the Po/Vm relation to more positive values by a few millivolts. Because of the exquisite sensitivity to cAMP (K1/2 = 0.75 µM) and the large effect of cAMP on Po (Gauss et al., 1998), modulation of HCN channels in sperm by cAMP is predicted to have profound functional consequences. Fourth, for the mammalian HCN4 channel expressed in heart and brain, it has been shown that a small but noticeable fraction of the current is carried by Ca2+ ions (Yu et al., 2004; Zhong et al., 2004). Because the pore motif is highly conserved among HCN channels, it is anticipated that sperm HCN channels are permeable for Ca2+ as well. Finally, HCN channels are often referred to as pacemakers, because they promote rhythmic electrical activity in neurons and cardiac myocytes (Pape, 1996). It needs to be examined in future studies whether these channels are also important for Ca2+ oscillations in sperm.

Model of the signaling pathway

Combining recent results and revised interpretations of previous findings, we arrive at a minimal model of the signaling pathway that controls Ca2+ entry in sperm from echinodermata (Fig. 1). Probably this model is also valid for other marine invertebrates. Binding of chemoattractant to receptor GC activates cGMP synthesis. The ensuing rise of cGMP activates K+-selective ion channels. The resulting hyperpolarization activates HCN channels. The depolarizing inward current through HCN channels in turn activates voltage-activated Cav channels. While the sequence of events is plausible, we emphasize that neither the cGMP-sensitive K+ channel nor the Cav channel(s) nor the mechanisms of their activation have been identified.

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Figure 1. Molecules involved in chemotactic signaling and their postulated function. Binding of the chemoattractant (ligand) to a membrane-bound guanylyl cyclase (GC) activates the synthesis of cGMP from GTP. Cyclic GMP opens cyclic nucleotide-gated (CNG) K+-se1ective channels, thereby causing hyperpolarization of the membrane. The cGMP signal is terminated by the hydrolysis of cGMP through phosphodiesterase (PDE) activity and inactivation of GC. On hyperpolarization, HCN channels allow the influx of Na+ (and perhaps also Ca2+) that leads to depolarization and thereby causes a rapid Ca2+ entry through voltage-activated Ca2+ channels (Cav), Ca2+ ions interact by unknown mechanisms with the axoneme of the flagellum and cause an increase of the asymmetry of flagellar beat and eventually a turn or bend in the swimming trajectory. Ca2+ is removed from the flagellum by a Na+/Ca2+ exchange mechanism.

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Our signaling scheme differs from previous models in some important respects. First, no initial decrease of [Ca2+]i is required to account for the swimming behavior of sperm in a gradient. Second, the relevant response of the cell is a rapid Ca2+ influx that causes a transient increase of the asymmetry of flagellar waveform. Third, the hyperpolarization is not involved in the changes in pHi and cAMP concentration. Therefore, pHi and cAMP do not directly participate in the opening of Cav channels.

Chemotactic signaling of sperm bears striking parallels to signaling in vision. In fact, the outer segment of rod photoreceptors originates from cilia-like structures. Like the sperm flagellum, the rod outer segment is a long compartment that harbors the signaling molecules. Sperm and rods register single molecules and photons, respectively, and the cellular response saturates when about 100 molecules or photons are caught (Pugh and Lamb, 2000; Kaupp et al., 2003). Cyclic GMP represents the intracellular messenger for both sperm and rods. However, the transduction mechanism is different. In sperm, the chemoattractant elicits a rise of cGMP concentration due to the synthesis by a receptor GC, whereas in rods, light absorption causes a decrease of cGMP concentration due to hydrolysis by PDE activity (Pugh and Lamb, 2000).

MOTOR RESPONSE AND NAVIGATION STRATEGY

  1. Top of page
  2. Abstract
  3. CHEMOATTRACTANTS AND THEIR RECEPTORS
  4. CELLULAR SIGNALING PATHWAY
  5. MOTOR RESPONSE AND NAVIGATION STRATEGY
  6. OUTLOOK
  7. Acknowledgements
  8. LITERATURE CITED

Early studies of chemotactic behavior in hydroids, tunicates, and mollusks revealed that the trajectory of sperm towards the source is characterized by alternation of tight loops and wide circular arcs (Miller and Brokaw, 1970; Miller, 1975; Miller, 1977; Miller, 1985). Wide arcs are made in the direction of the source, while tight loops occur when sperm swim away from it. In a defined gradient, generated by the release of the chemoattractant from a caged compound, sperm show exactly the same behavior as described in those early studies (Böhmer et al., 2005).

This behavior together with the Ca2+ dependence of the flagellar waveform (see above) inspired a simple concept: continuous stimulation of sperm while swimming up gradient keeps [Ca2+]i low and the flagellar beat symmetrical. If a sperm cell diverts from its course and senses a decrease of chemoattractant, [Ca2+]i goes up, the flagellar beat becomes asymmetrical, and the cell makes a turn (Miller, 1985; Cook et al., 1994). A corollary of this concept is that the swimming pattern—straight versus turn—signifies the swimming direction with respect to the direction of the gradient.

As perspicuous as this concept is, it does not account for several observations. First, on stimulation by the chemoattractant or by the intracellular release of cyclic nucleotides, [Ca2+]i rises rather than decreases (Kaupp et al., 2003; Solzin et al., 2004; Böhmer et al., 2005; Wood et al., 2005). Second, the first detectable motor response is an increase of the asymmetry of the flagellar beat and hence a turn or bend in the otherwise circular trajectory. Initial straight segments in the swimming trajectories—before the turn—have not been observed (Kaupp et al., 2003; Böhmer et al., 2005). Third, the model does not take into account the substantial delay of the Ca2+ influx and hence of the motor response (Kaupp et al., 2003). Fourth, the model ignores the periodic nature of the trajectory: turns and relatively straight sections follow in regular sequels. Specifically, the model does not explain why sperm should bear away once they are on the right track. As long as the concentration of the chemoattractant is increasing and [Ca2+]i is kept low, according to this model, sperm should head for the source on a curvilinear path.

How can we reconcile these seemingly contradicting observations and arrive at a coherent model? Periods of straighter swimming always follow chemotactic turns even when sperm are stimulated by cGMP or by a homogenous concentration of chemoattractant, that is, the pattern is independent of a chemical gradient (Kaupp et al., 2003). We have defined this inherent pattern as a motor response unit or “turn-and-run” (Böhmer et al., 2005). A new model that takes into account the motor response unit as an essential feature of navigation is shown in Figure 2a. Sperm “measure” the increasing concentration of chemoattractant during a “sampling period” when swimming up gradient. After a delay of several hundreds of milliseconds, a Ca2+ spike is generated that gives rise to a turn. The delay corresponds to almost half a circle and coincides with the period of swimming down gradient. This is the reason why turns seem to be the result of swimming down gradient. During the following period of straighter swimming up gradient, the next sampling of chemoattractant occurs. By this means, sperm swim in circles that are displaced (by epicycloid-like movement) in the direction of the source of the chemoattractant.

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Figure 2. Scheme of the navigation strategy of sperm in a gradient of chemoattractant. a, In a gradient of chemoattractant (background), a sperm cell (black), swimming on a circular trajectory, detects an increase of the chemoattractant as long as it moves up-gradient (“sampling phase”, orange trace). Sampling is followed by a delay phase when sperm continue to swim on a circle down-gradient (green trace, Φ of about 160°, corresponding to a delay of about 300 ms). After the delay, a Ca2+ spike occurs. As a consequence, the curvature of the trajectory transiently increases (“turn”) and thereafter decreases (“run”) below the original value (red arrow). The width of the arrow indicates the range of angles at which sperm leave the circle. Moving again up-gradient, the cell is passing through the next sampling period (after (Böhmer et al., 2005). b, Presumed trajectory of a freely swimming sperm during chemotaxis. Several repetitions of the response unit (“turn and run”) direct the cell on a curved helical path towards the egg.

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Stringing together response units by itself would not result in directed movement. The proposed model requires precise timing between sampling, Ca2+ spike, and turn. In fact, Ca2+ spikes are synchronized with the stimulus function, that is, the concentration profile along the swimming path (Böhmer et al., 2005). The concept of a sampling period implies that sperm probe their environment intermittently rather than continuously. Consequently, sperm must be endowed with a mechanism that effectively resets the system after every Ca2+ spike or turn. Reactions that terminate signaling could be inactivation of GC and Cav channels, hydrolysis of cGMP, and removal of Ca2+ by Na+/Ca2+ exchange.

A decrease of the [Ca2+]i may not be the only factor that reduces the curvature of the swimming path after a turn, because the curvature commences to relax below pre-stimulus values when [Ca2+]i is still above resting levels (Böhmer et al., 2005; Wood et al., 2005). During the “run” period, the swimming speed of sperm cells is significantly enhanced (Kaupp et al., 2003). The speed is negatively correlated with the curvature of the trajectory and speeding contributes to the straightening of the trajectory (Böhmer et al., 2005). In Ca2+-free seawater, that is, under conditions that abolish both Ca2+ influx and turns, cells still speed up on stimulation and the curvature decreases. Interestingly, the delay of this response is of the same range as the delay of the Ca2+ signal and the motor response in the presence of Ca2+ (Kaupp et al., 2003; Böhmer et al., 2005). This observation suggests that cGMP triggers a yet unknown mechanism independent of Ca2+ entry.

Chemotaxis has been extensively studied in bacteria and comparison with sperm chemotaxis may provide important mechanistic insights. Bacteria alternate between two behavioral modes—tumbling and run. The runs directed up gradient are longer than those directed down gradient. A consequence of this behavior is that bacteria increase the time moving towards the source and decrease the time moving away from it (Eisenbach et al., 2004). The wide arcs or periods of straight swimming of sperm are reminiscent of the bacterial runs, while the tight loops or turns might resemble the tumbling mode of bacteria. However, this analogy is superficial because there is a fundamental difference. Bacteria take random directions after tumbling, whereas the wide arcs of sperm show a pronounced tendency towards the source. Thus, bacteria approach the source by a biased random walk, whereas the path directions of sperm are non-random.

Due to their small size, bacteria sense chemoattractants by comparing their concentrations at two consecutive moments (“temporal sensing”) (Macnab and Koshland, 1972; Eisenbach et al., 2004). A sperm cell is sufficiently long (∼50 µm) to allow comparison of concentrations at different sites along the flagellum (“spatial sensing”). However, stimulation with a homogenous field of chemoattractant evokes a chemotactic response (“turn-and-run”) (Kaupp et al., 2003), suggesting that sperm also use a temporal sensing mechanism.

Bacteria register both ascending and descending gradients of chemotactically active substances by means of the occupancy level of their receptors (for reviews see Eisenbach et al., 2004). The dissociation constants KD of most bacterial chemoattractants are in the micro- to millimolar range. Therefore, bound and free ligands are in chemical equilibrium on a sub-millisecond time scale. In contrast, bound and free chemoattractants of sperm are not in chemical equilibrium for the following considerations. The KD values are extremely low, that is, in the picomolar range (Nishigaki and Darszon, 2000), and bound ligands are not expected to dissociate from the receptor during a motor response. Furthermore, the number of molecules that hit a flagellum within 1 sec is roughly N = 18 at a concentration of 1 pM (Böhmer et al., 2005). Assuming a KD value of 1 pM and a receptor density of ca. 20,000 per flagellum (Shimomura and Garbers, 1986; Shimizu et al., 1994; Nishigaki and Darszon, 2000), it would take almost 20 min to reach chemical equilibrium. This notion has two important functional consequences. First, the chemotactic response is controlled by the rate of chemoattractant binding rather than by the level of occupancy. Second, sperm may be unable to sense a descending gradient of the chemoattractant, because bound ligands do not dissociate from the receptor. Thus, the physiological important parameter may be the fraction of receptors that are active at a time rather than the fraction of occupied receptor.

OUTLOOK

  1. Top of page
  2. Abstract
  3. CHEMOATTRACTANTS AND THEIR RECEPTORS
  4. CELLULAR SIGNALING PATHWAY
  5. MOTOR RESPONSE AND NAVIGATION STRATEGY
  6. OUTLOOK
  7. Acknowledgements
  8. LITERATURE CITED

While a coherent picture of the excitatory signaling events is emerging, the mechanisms that terminate signaling and adjust the excitability of the cell are largely unknown. In order to escape saturation of both the receptor occupancy and the Ca2+ response, the cell must dispose of mechanisms that regulate its sensitivity. While swimming up gradient, sperm probably continuously adapt to the ever-increasing concentration of the chemoattractant. A comprehensive model of chemotaxis must, therefore, incorporate the interplay between excitation, adaptation, and recovery.

When spatially restricted in the experimental chamber, sperm glide at the glass–water interface and, therefore, move in circles in the observation plane. We should emphasize that our model has been derived from the two-dimensional movement of sperm under conditions of this kind. Freely swimming sperm move on a three-dimensional helical trajectory (Crenshaw, 1989). We propose that the two-dimensional epicycloid-like trajectories that are observed under the microscope reflect a helical path that becomes gradually bent towards the source of the chemoattractant (Fig. 2b). In this sense, sperm display “true” chemotaxis, that is, directed movement toward the source. However, it needs to be shown how sperm keep on their path once the axis of the helix has become oriented towards the source.

Acknowledgements

  1. Top of page
  2. Abstract
  3. CHEMOATTRACTANTS AND THEIR RECEPTORS
  4. CELLULAR SIGNALING PATHWAY
  5. MOTOR RESPONSE AND NAVIGATION STRATEGY
  6. OUTLOOK
  7. Acknowledgements
  8. LITERATURE CITED

This work was supported by the Deutsche Forschungsgemeinschaft and Fonds der chemischen Industrie.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. CHEMOATTRACTANTS AND THEIR RECEPTORS
  4. CELLULAR SIGNALING PATHWAY
  5. MOTOR RESPONSE AND NAVIGATION STRATEGY
  6. OUTLOOK
  7. Acknowledgements
  8. LITERATURE CITED