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Ligands and receptors mediating signal transduction in sea urchin spermatozoa

Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093-0202, USA

Correspondence should be addressed to A T Neill; Email: aneill{at}ucsd.edu

	Abstract

Top Abstract Introduction Activation of motility Chemokinesis and chemotaxis Acrosome reaction Future directions References

 
Sea urchins have long been a model system for the study of fertilization. Much has been learned about how sea urchin sperm locate and fertilize the egg. Sperm and eggs are spawned simultaneously into the surrounding seawater. Sperm signaling pathways lead to downstream events that ensure fertilization. Upon spawning, sperm must acquire motility and then they must swim towards or respond to the egg in some way. Finally, they must undergo a terminal exocytotic event known as the acrosome reaction that allows the sperm to bind to the vitelline layer of the egg and then to fuse with the egg plasma membrane. Motility is stimulated by exposure to seawater, while later events are orchestrated by factors from the egg. The sperm signaling pathways are exquisitely tuned to bring the sperm to the egg, bind, and fuse the two cells as quickly as possible.

	Introduction

Top Abstract Introduction Activation of motility Chemokinesis and chemotaxis Acrosome reaction Future directions References

 
Sea urchins are marine invertebrates found at the base of the deuterostome lineage that leads to the vertebrates. They have separate sexes and are external spawners. The sea urchin is an excellent model system for the study of fertilization because large quantities of easily obtainable gametes are readily available. Much has been learned about gamete interactions and signal transduction pathways involved in sea urchin fertilization. This review will focus on what is known about signal transduction pathways in sea urchin spermatozoa that allow the sperm to respond to the extracellular environment and to find and fertilize the egg.

Sperm are terminally differentiated cells that serve three major functions: to deliver the centrosome to the egg, to metabolically activate the dormant egg, and to restore the 2N genome. In order to fertilize an egg, a spermatozoon must undergo several important steps. First, the spermatozoon must gain motility so that it can travel a distance to the egg. Secondly, it must be stimulated by or attracted to the egg. Finally, the spermatozoon needs to undergo changes that allow it to bind to and fuse with the egg plasma membrane. In sea urchins, motility is activated when sperm are spawned into seawater. As they approach the egg, sperm are stimulated by factors associated with a jelly coat that surrounds the egg. Small peptide signaling molecules can have either a chemokinetic or chemotactic effect on sperm. Chemokinesis is an increase in or activation of motility in response to some signal while chemotaxis actually involves the sperm turning in the direction of the egg. Both chemokinesis and chemotaxis have been described for sea urchin sperm. Once the sperm has advanced close enough to the egg, carbohydrate signaling molecules induce the sperm to undergo the exocytotic acrosome reaction (AR). The AR exposes sperm molecules that allow the sperm membrane to bind to and ultimately fuse with the egg membrane. Figure 1 is a representation of the events that occur during sea urchin fertilization.

View larger version (37K): [in this window] [in a new window]   Figure 1 Sea urchin fertilization. When sperm are spawned into seawater they become motile and begin to swim (1). Small peptides from the egg jelly coat serve either to further activate motility and/or to attract the sperm via chemotaxis (2). When the sperm has come into close contact with the egg, a polysaccharide from the jelly coat induces the AR (3). During the AR, sperm undergo a number of ionic fluxes (occurring at 3a) that lead to exocytosis of the acrosomal vesicle (3b) and polymerization of actin to form the acrosomal process that binds the sperm to the vitelline layer of the egg (3c). The bindin-coated membrane of the process then fuses with the egg plasma membrane (3d). N, nucleus; M, mitochondrion.

	Activation of motility

Top Abstract Introduction Activation of motility Chemokinesis and chemotaxis Acrosome reaction Future directions References

In addition to lowering the CO₂ tension, spawning may also cause a hyperpolarization of the sperm plasma membrane. External potassium ([K⁺]_o) is higher in semen than it is in seawater (Christen et al. 1986), and the transition from high [K⁺]_o in the testis to lower [K⁺]_o in seawater may result in plasma membrane hyperpolarization. Sperm activation is inhibited when [K⁺]_o is 100 mM (Darszon et al. 1999), suggesting that the transition from high to low [K⁺]_o is critical to activation ([K⁺]_o in seawater is 10 mM). Hyperpolarization could activate the flagellar voltage-dependent Na⁺/H⁺ exchanger and thus contribute to the rise in pH_i (Lee, 1984a,b). An Na⁺-K⁺-ATPase may also be critical for keeping intracellular sodium ([Na⁺]_i) low and thus contributing to intracellular pH (pH_i) regulation (Gatti & Christen 1985).

Hyperpolarization could contribute to activation both by participating in the pH_i rise and by activating adenylyl cyclase (AC). Sea urchin sperm AC is activated by membrane potential (Bookbinder et al. 1990, Beltran et al. 1996). If a hyperpolarization event activates AC, then the concomitant rise in cAMP may activate a cAMP-dependent protein kinase. Phosphorylation of proteins in the flagellar axoneme may be crucial for the initiation of motility (Garbers 1989, Morisawa 1994).

	Chemokinesis and chemotaxis

Top Abstract Introduction Activation of motility Chemokinesis and chemotaxis Acrosome reaction Future directions References

 
Sperm-activating peptides (SAPs) are small peptides (10–15 amino acids) isolated from the jelly coat of sea urchin eggs. These peptides are diffusible and interact with sperm at some distance from the egg. When they bind to sperm, SAPs cause a cellular activation, resulting in either a chemokinetic or chemotactic response and they often act in a species-specific manner. They may also play a role in induction of the AR (see Acrosome reaction below). Approximately 75 SAPs have been described from over 15 species of sea urchins (Suzuki 1995).

The first SAP to be purified and characterized was the decapeptide speract (also known as SAP-1). Speract, which is derived from the egg jelly of Strongylocentrotus purpuratus and other species (Suzuki 1995), induces sperm phospholipid metabolism, respiration, and motility at picomolar concentrations at an extracellular pH (pH_o) of 6.6 (Kopf et al. 1979, Garbers & Kopf 1980, Hansbrough et al. 1980). In normal seawater at pH 8.0, speract induces a number of changes in sperm, including Na⁺ and Ca²⁺ influx, K⁺ and H⁺ efflux, and increases in the concentrations of cAMP and cGMP (Darszon et al. 1999, 2001).

In S. purpuratus, speract elicits a sperm response by binding to a 77 kDa plasma membrane receptor (Dangott & Garbers 1984, Dangott et al. 1989) which is localized exclusively in the sperm flagellum (Cardullo et al. 1994). Binding of speract to its receptor activates a membrane guanylyl cyclase (GC) (Bentley et al. 1988, Garbers 1989). Activation of GC results in an increase of cGMP, which in turn opens a cGMP-dependent K⁺ channel, leading to the hyperpolarization of the plasma membrane (Babcock et al. 1992, Galindo et al. 2000). Blocking of this hyperpolarization with high [K⁺]_o abolishes all effects of speract except for the initial increase in cGMP (Harumi et al. 1992). The hyperpolarization of the membrane activates at least three proteins.

The first of these voltage-dependent molecules to be studied was the voltage-dependent Na⁺/H⁺ exchanger (Lee & Garbers 1986). When the sperm hyperpolarize during K⁺ efflux, this exchanger is activated and pH_i increases. The increase in pH_i feeds back on the original response via pH-sensitive phosphatases and phosphodiesterases that dephosphorylate (inactivate) GC and decrease cGMP levels (Ramarao & Garbers 1985, Ward et al. 1985b, Garbers 1989).

[Na⁺]_i increases from 20 mM to 35 mM during the speract response; however, not all of this increase can be attributed to Na⁺/H⁺ exchange since the pH_i increase is saturated before [Na⁺]_i saturates. Blocking of Na⁺/Ca²⁺ and K⁺-dependent Na⁺/Ca²⁺ exchangers does not alter the kinetics of [Na⁺]_i fluxes, indicating that these types of channels are not directly involved in the speract response (Rodriguez & Darszon 2003). However, activity of a flagellar K⁺-dependent Na⁺/Ca²⁺ exchanger is required for sperm motility, presumably to maintain low [Ca²⁺]_i (Su & Vacquier 2002). The other channel responsible for Na⁺ influx during the speract response remains unknown.

Another molecule that is activated in response to the speract-induced membrane hyperpolarization is AC (Beltran et al. 1996). Activation of AC leads to an increase in cAMP, which may participate in the opening of a Ca²⁺ channel that transiently increases intracellular calcium ([Ca²⁺]_i) (Cook & Babcock 1993). The influx of Ca²⁺ causes a transient membrane depolarization that follows the hyperpolarization due to K⁺ efflux (Beltran et al. 1996). Voltage-dependent Ca²⁺ channel blockers fail to affect the speract-induced Ca²⁺ influx, indicating that the channel involved is not directly modulated by voltage (Rodriguez & Darszon 2003). Stopped-flow techniques have been used to establish that the pH_i increase induced by speract hyperpolarization precedes the Ca²⁺ influx by 120 ms (Nishigaki et al. 2001).

The increase in cAMP coupled with the membrane hyperpolarization leads to the activation of a hyperpolarization-activated and cyclic nucleotide-gated channel (HCN) known as SPIH (Gauss et al. 1998). HCN channels are known to control rhythmic firing of neurons and pacemakers in the heart (Robinson & Siegelbaum 2003). The channels are poorly selective for K⁺, allowing both K⁺ and Na⁺ influx at a ratio of 5:1; channels with such properties have been measured in sea urchin sperm flagellar membranes (Labarca et al. 1996, Sanchez et al. 2001). Because it may be involved in the establishment of periodicity, it has been suggested that the SPIH channel could modulate flagellar beating and thus contribute to a chemotactic response (Kaupp & Seifert 2001). Indeed, a rhythmic pattern of Ca²⁺ increases has been observed in sperm flagella in response to speract (Wood et al. 2003), but a chemotactic response to speract has yet to be demonstrated. Figure 2 provides a general scheme of the signaling pathway activated by binding of speract.

View larger version (32K): [in this window] [in a new window]   Figure 2 The speract signaling pathway. Binding of speract to its receptor stimulates GC and increases cGMP (1). A cGMP-dependent K+ channel opens (2), hyperpolarizing the sperm membrane. This hyperpolarization mediates the activation of downstream proteins. An Na+/H+ exchanger activates, resulting in an increase in pHi (3). AC is activated (4), producing cAMP that stimulates the opening of a Ca2+ channel. Hyperpolarization and cAMP also stimulate the activity of the SPIH channel (5), which may control the rhythmic nature of flagellar beating.

	Acrosome reaction

Top Abstract Introduction Activation of motility Chemokinesis and chemotaxis Acrosome reaction Future directions References

 
First described in the early 1950s by Jean Clark Dan, the AR is a crucial step in fertilization that occurs when sperm contact the jelly coat surrounding the egg (Dan 1952, 1954a, 1967). Both extracellular Ca²⁺ and Na⁺ are required for the AR (Dan 1954b, Schackmann & Shapiro 1981) which is characterized by two major physiological events: the exocytosis of the acrosomal vesicle and the extension of the acrosomal process. Acrosomal exocytosis releases the protein bindin (Vacquier & Moy 1977, Vacquier et al. 1995, Zigler & Lessios 2003), which mediates the species-specific adhesion of sperm to egg (Glabe & Lennarz 1979). The acrosomal process is formed by the pH_i-dependent polymerization of actin (Tilney et al. 1978). The process extends 1 µm from the tip of the sperm head, and is covered by the bindin-coated membrane that will fuse with the egg plasma membrane (Barre et al. 2003) (Fig. 1). The interaction between the plasma membranes of sperm and egg is a receptor-mediated event, with the egg receptor for bindin recognizing and binding species specifically to sperm bindin (Kamei & Glabe 2003).

The ligand from egg jelly that binds sperm and induces the AR in S. purpuratus is a fucose sulfate polymer (FSP) (SeGall & Lennarz 1979, Vacquier & Moy 1997). Egg jelly sulfated polysaccharides from a number of species have been isolated and characterized, and all induce the AR species specifically (Alves et al. 1997). The species specificity of this interaction is determined by the glycosidic linkage of the polymer and the pattern of sulfation of the sugar residues (Hirohashi et al. 2002, Vilela-Silva et al. 2002). Interestingly, the ligand that induces the AR in sea urchins is a pure polysaccharide, with no associated protein (Vacquier & Moy 1997).

Within seconds, binding of FSP induces ion fluxes; Na⁺ and Ca²⁺ influx, while K⁺ and H⁺ efflux (Darszon et al. 1999, 2001). These ion fluxes result in changes in membrane potential (Schackmann et al. 1981, Gonzalez-Martinez & Darszon 1987), an increase in [Ca²⁺]_i (Guerrero & Darszon 1989b), and an Na⁺-dependent increase in pH_i of 0.25 units (Lee et al. 1983, Guerrero & Darszon 1989b). Binding of FSP also leads to a number of other physiological changes: a tenfold increase in inositol 1,4,5-trisphosphate (IP₃) (Domino & Garbers 1988), a Ca²⁺-dependent activation of AC (Watkins et al. 1978) that leads to an increase in cAMP (Garbers & Kopf 1980), and increases in the activities of protein kinase A (Garbers et al. 1980, Porter & Vacquier 1986, Garcia-Soto et al. 1991), phospholipase D (Domino et al. 1989), and nitric oxide synthase (Kuo et al. 2000). It is interesting to note that the same physiological changes associated with motility events (fluxes in membrane potential, Ca²⁺ influx, and an increase in pH_i) also drive the AR. Perhaps spatial and temporal patterns of these events dictate their signaling specificity.

In S. purpuratus, FSP binds to the sea urchin receptor for egg jelly (suREJ1) (Moy et al. 1996). Antibodies directed against this protein show that it is localized along the length of the flagellum and also as a thin band at the tip of the sperm head, overlying the acrosomal vesicle (Trimmer et al. 1985). Additionally, some monoclonal antibodies directed against suREJ1 induce Ca²⁺ influx and the AR (Trimmer et al. 1986, Moy et al. 1996). suREJ1 is a homolog of the human polycystic kidney disease protein polycystin-1 (Moy et al. 1996). Polycystin-1 and polycystin-2 associate to form unique non-selective cation channels (Hanaoka et al. 2000, Xu et al. 2003).

In addition to suREJ1, two other polycystin-1 homologs have been cloned and shown to be present in sea urchin sperm: suREJ2 (Galindo et al. 2003) and suREJ3 (Mengerink et al. 2002). While suREJ2 does not appear to be involved in the AR, suREJ3 has many attributes that suggest it may participate in AR signaling. Unlike suREJ1, which only contains one putative transmembrane segment and a short cytoplasmic tail, suREJ3 is a larger protein that contains all 11 putative transmembrane segments of human polycystin-1 (Mengerink et al. 2002). This includes the C-terminal transmembrane region that is homologous to voltage-dependent Ca²⁺ channels and has been implicated in associations with polycystin-2 (Qian et al. 1997, Xu et al. 2003). Interestingly, sea urchin sperm possess a polycystin-2 homolog (suPC2), and suREJ3 and suPC2 are physically associated in the sperm plasma membrane (Neill et al. 2004). Both suREJ3 and suPC2 localize exclusively as a thin band on the plasma membrane overlying the acrosomal vesicle (Mengerink et al. 2002, Neill et al. 2004). Also, channel activities with properties similar to polycystin channels have been measured from sea urchin sperm membranes (Lievano et al. 1990, Beltran et al. 1994). This evidence suggests that REJ and polycystin proteins may be directly participating in the ion fluxes that follow binding of FSP and that lead to the AR. This is an interesting parallel between invertebrate fertilization and human disease proteins.

When FSP binds to Lytechinus pictus sperm, it induces a transient hyperpolarization followed by a membrane depolarization (Gonzalez-Martinez & Darszon 1987). These membrane potential changes are most likely occurring in S. purpuratus sperm as well; when [K⁺]_o is raised from 10 mM to 40 mM, the Ca²⁺ increase and AR are inhibited (Schackmann et al. 1978), as is the increase in pH_i (Guerrero & Darszon 1989b). The Na⁺ dependence of the increase in pH_i suggests a role for hyperpolarization-activated Na⁺/H⁺ exchange (Gonzalez-Martinez et al. 1992). However, this Na⁺/H⁺ exchange is probably not mediated by the same pathway as the speract-induced Na⁺/H⁺ exchange, because in the AR this exchange is Ca²⁺ dependent (Guerrero et al. 1998), while in the speract response it is not (Schackmann & Chock 1986). Even if Na⁺/H⁺ exchange is involved in Na⁺ influx, [Na⁺]_i saturates well after pH_i saturates, implying that another channel is involved in the [Na⁺]_i increase (Rodriguez & Darszon 2003).

The Ca²⁺ influx that occurs in response to FSP has two distinct phases; the influxes associated with these phases occur through separate channels (Guerrero & Darszon 1989b). FSP binding triggers the opening of the first channel, which is Ca²⁺ selective, blocked by verapamil and dihydropyridines, and inactivates after opening. The second channel opens 4 s later, is sensitive to Ni²⁺, insensitive to verapamil and dihydropyridines, is permeable to Mn²⁺, and does not inactivate, but produces a sustained Ca²⁺ influx. The first channel will open even if the pH_i increase is blocked, but the second channel will not. If the opening of the first channel is blocked, the second channel will not open (Guerrero & Darszon 1989a,b). Thus, the operation of these two channels is physiologically linked even though they represent distinct modes of Ca²⁺ entry.

Opening of both channels is required for the AR (Darszon et al. 1999, Hirohashi & Vacquier 2002b). The second channel alone can be opened by a lower molecular weight hydrolyzed form of FSP (hFSP), but the AR does not take place (Hirohashi & Vacquier 2002b). hFSP does cause an increase in pH_i, further indicating that a rise in pH_i is an important signal for the second channel to open.

Increasing evidence indicates that the second Ca²⁺ channel is a store-operated Ca²⁺ channel (Gonzalez-Martinez et al. 2001, Hirohashi & Vacquier 2003). The increase in IP₃ (Domino & Garbers 1988) that occurs in response to FSP, coupled with the fact that IP₃ receptors have been detected in sea urchin sperm (Zapata et al. 1997), suggest that this signaling system may function during the AR. IP₃-mediated release of Ca²⁺ from intracellular stores is a crucial step in store-operated Ca²⁺ entry (Putney et al. 2001). Although sperm lack an endoplasmic reticulum, it has been suggested that the acrosomal vesicle may be acting as the intracellular Ca²⁺ store (Gonzalez-Martinez et al. 2001). In sea urchins, opening of the store-operated Ca²⁺ channel alone is sufficient to trigger acrosomal exocytosis, but not for a complete AR (no acrosomal process is formed) (Hirohashi & Vacquier 2003). Figure 3 provides an illustration of the store-operated calcium influx associated with the AR.

View larger version (49K): [in this window] [in a new window]   Figure 3 Store-operated Ca2+ entry during the AR. Binding of FSP to suREJ1 (1) leads to the opening of the first Ca2+ channel. Phospholipase activity increases, resulting in an increase in IP3 (2). IP3 binds its receptor, perhaps located in the acrosomal membrane, releasing Ca2+ from this internal store (3). The Ca2+ released from the store stimulates opening of the second Ca2+ channel via some as yet unknown mechanism, resulting in a large, sustained increase in intracellular Ca2+ (5). Also important for operation of the second channel is an increase in pHi mediated, in part, by an Na+/H+ exchanger (4).

Two additional channels have been detected that contribute to the AR. Tetraethylammonium (TEA⁺), which is a blocker of K⁺ channels, inhibits the egg jelly-induced AR (Schackmann 1989) and TEA⁺-sensitive K⁺ channel activities have been measured from sperm membranes (Lievano et al. 1985). Additionally, the anion channel blocker 4,4'-diisothiocyanostilbene disulphonic acid (DIDS) blocks the AR and a DIDS-sensitive Cl^- channel activity is present in sperm plasma membranes. This Cl^- channel may be important either to maintain the membrane potential prior to AR induction or to contribute directly to ion fluxes during the AR (Morales et al. 1993).

	Future directions

Top Abstract Introduction Activation of motility Chemokinesis and chemotaxis Acrosome reaction Future directions References

 
While much is known about signal transduction in sea urchin spermatozoa, many questions remain. Table 1 contains a list of all of the sperm proteins described in this review that have a measured or proposed function in signal transduction. Of the 25 proteins described, only about one-third have been cloned. Additionally, the ligands or activators that stimulate many of the proteins are unknown.

	References

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