Molecular Reproduction & Development 77:739–750 (2010)
The Physiological Acquisition of Amoeboid Motility in
Nematode Sperm: Is the Tail the Only Thing the Sperm Lost?
JUAN J. FRAIRE-ZAMORA AND RICHARD A. CARDULLO*
Department of Biology and the Graduate Program in Evolution, Ecology, and Organismal Biology, University of California, Riverside, California
Nematode spermatozoa are highly specialized amoeboid cells that must acquire motility through the extension of a single pseudopod. Despite morphological and To acquire motility, a spherical molecular differences with ！agellated spermatozoa (including a non-actin-based spermatid must ，rst undergo [a] cytoskeleton), nematode sperm must also respond to cues present in the female process . . . in which a pseudopod reproductive tract that render them motile, thereby allowing them to locate and fertilize is extended, conferring motility the egg. The factors that trigger pseudopod extension in vivo are unknown, although to the cell in an amoeboid current models suggest the activation through proteases acting on the sperm surface fashion. resulting in a myriad of biochemical, physiological, and morphological changes. Compelling evidence shows that pseudopod extension is under the regulation of physiological events also observed in other eukaryotic cells (including ！agellated sperm) that involve membrane rearrangements in response to extracellular cues that initiate various signal transduction pathways. An integrative approach to the * Corresponding author: study of non！agellated spermatozoa will shed light on the identi，cation of unique Department of Biology, University of and conserved processes during fertilization among different taxa. California, Riverside, CA 92521. E-mail: firstname.lastname@example.org
Mol. Reprod. Dev. 77: 739–750, 2010. ß 2010 Wiley-Liss, Inc.
Published online 19 May 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.21193 Received 5 October 2009; Accepted 22 March 2010
INTRODUCTION universal, careful reviews reveal that many taxa produce
non！agellated sperm (Morrow, 2004; see Fig. 1A). Fertilization is common to all multicellular organisms that Perhaps, the most studied taxonomic group with motile reproduce sexually. In this process, two gametes, typically non！agellated sperm is the Phylum Nematoda. This phylum haploid cells, fuse to form a new individual. In order for is extremely diverse with both free-living and parasitic re- successful fertilization to occur, two events are necessary. presentatives comprising gonochoristic, hermaphroditic, First, the gametes must ，nd one another so that they come and parthenogenetic reproductive strategies (Poinar, into direct physical contact. Second, upon sperm–egg bind- 1983). Morphologically, the spermatozoa in the entire group
are characterized by the absence of a ！agellum and an ing, a complex series of biochemical and physiological
events must occur, culminating in fusion and egg activation acrosome as well as the presence of membranous vesicles (Yanagimachi, 1994). (Justine, 2002). To acquire motility, a spherical spermatid The ‘‘typical’’ fertilization process involves a sessile egg must ，rst undergo the process known as spermiogenesis or that is encountered by a highly motile spermatozoon. Most
frequently, the spermatozoon is propelled by a microtubule-
based ！agellum that brings the gametes into suf，ciently Abbreviations: MSP, major sperm protein; MO, membranous organelle; close contact to ultimately result in fertilization. Although MPOP, MSP polymerization organizing protein; SOCE, store operated calcium this general view of fertilization is often assumed to be entry; TEA, triethanolamine; VDX, vas deferens extract.
ß 2010 WILEY-LISS, INC.
Molecular Reproduction & Development FRAIRE-ZAMORA AND CARDULLO
Figure 1. Not everyone needs a tail to get around. Species with non！agellated spermatozoa are more abundant in nature than previously realized, opening new venues for physiological comparisons among sperm from different taxa. A: In the Eumetazoan lineage, non！agellated sperm are not uncommon. The phylogenetic tree shows Phyla that contain at least one species bearing non！agellated spermatozoa (in red); Phyla containing only non！agellated spermatozoa (in blue); and Phyla containing only ！agellated spermatozoa (in black) (Phylogenetic tree modi，ed from the Tree of Life Web project http://www.tolweb.org/, with information from Marrow, 2004). B: The Phylum Nematoda is representative of a taxonomic group where all the members possess motile amoeboid spermatozoa. Male gametes in this group lack a ！agellum and an acrosome, and are characterized by the presence of Membranous Organelles that fuse to the membrane during acquisition of motility. Prior to pseudopod extension the cell must undergo physiological and morphological changes that include membrane rearrangements, extension of ，llopodia-like projections (spikes), and the con，nement of organelles into the cell body, while a pH gradient is formed from the tip to the base of the pseudopod.
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OTILITY IN NEMATODE SPERM M
zoa can crawl in vitro at an average instantaneous velocity of sperm activation, in which a pseudopod is extended, con-
ferring motility to the cell in an amoeboid fashion (see 12.0 4.9 mm/min (Royal et al., 1997), similar to the reported Fig. 1B). This process of acquisition of motility has been in vivo average velocity of 8.07 0.67 mm/min of male sper- most extensively studied in the nematodes Ascaris suum matozoa crawling in the hermaphrodite uterus (Kubagawa et and Caenorhabditis elegans, and is currently thought to be al., 2006). Protease-activated spermatozoa from C. elegans the general mode of sperm locomotion in all nematodes. are also unable to crawl.
Although, most studies on these sperm have been per-
formed using molecular genetics and biochemistry, an inte- PLASMA MEMBRANE DYNAMICS AND grative physiological framework is lacking. The present MICRODOMAINS study is intended to summarize the physiological pathways
that lead to the in vitro extension of the pseudopod in The ，rst step in the process of sperm activation is the nematode sperm from both Ascaris and Caenorhabditis. rearrangement of the plasma membrane. Roberts and Ward (1982b) observed this dramatic rearrangement by attaching
latex beads to the plasma membrane of C. elegans sper- IN VITRO PSEUDOPOD EXTENSION AND matids and, using a microscope, followed their movements CRAWLING OF NEMATODE SPERMATOZOA throughout the activation process. This experiment revealed Nematode spermatozoa initiate motility subsequent to that the plasma membrane of spermatids undergoes an the extension of the pseudopod (in the case of C. elegans, or intermittent, nondirected movement at a rate of 10–15 mm/ lamellipod in A. suum) allowing the cell to crawl on a min on discrete portions of the cell surface. This movement, substrate. A peculiarity in these amoeboid cells is that they coupled to cell rotation, initiates after treatment with Mon- contain non-actin-based micro，laments responsible for ensin, stops once the pseudopod is completely formed, and pseudopod extension (Nelson et al., 1982). Instead of actin, is not present in mutant sperm that fail to extend a pseudo- nematode sperm form ，laments from the Major Sperm pod (Roberts and Ward, 1982b). Protein (MSP), a 14 kDa protein that constitutes 15% of the The extension of ‘‘spike’’ structures in nematode sperm total protein in the sperm (Klass and Hirsh, 1981). To coincides with membrane rearrangements and a highly ！uid polymerize, MSP must be present in dimers to elongate cell surface, resulting in the dynamic steps of protrusion, ，laments and ，bers allowing the cell to crawl (Roberts and retraction, thickening, and fusion of spikes to coalesce into Stewart, 2000). a pseudopod or lamellipod (Shakes and Ward, 1989; Rodri- Despite the threefold difference in maximum length be- guez et al., 2005). After the extension of the pseudopod and tween the spermatozoa of C. elegans ( 9 mm) and A. suum before the acquisition of motility, the nematode-speci，c Mem-
( 26 mm; Royal et al., 1997), the process of pseudopod branous Organelles (MOs) localize to the periphery of the cell extension in both nematodes is very similar and initiates with body and fuse to the plasma membrane, delivering proteins rearrangements of the plasma membrane (Nelson and Ward, and membrane material necessary for the total extension of 1980) and the extension of transient membrane protrusions the pseudopod, motility acquisition and successful fertiliza- known as ‘‘spikes’’ in C. elegans or ，lipodia in A. suum. tion (L’Hernault, 2006; Singson et al., 2008). At the time of Filaments of MSP form these ‘‘spikes,’’ which precede the MOs fusion, all the membrane rearrangements cease in the formation of the pseudopod in both A. suum and C. elegans cell body while the initiation of directed membrane ！ow from (Shakes and Ward, 1989; Rodriguez et al., 2005). Thus, for the tip to the base of the pseudopod confers motility to the cell the purpose of this review, we refer to the physiological (Roberts and Ward, 1982a). Protein insertion has been signals that initiate membrane rearrangements and spike pro- observed at the leading edge of the pseudopod, although it trusions as sperm activation, a process that results in pseu- is not clear how this process occurs as there are no organelles dopod extension and the consequent acquisition of motility. found in this region and no endocytosis processes or vesicle Although the molecule(s) responsible for in vivo sperm traf，cking has been reported (Roberts and Ward, 1982a; activation in nematodes are not known to date, the process Pavalko and Roberts, 1987; Shakes and Ward, 1989). of in vitro sperm activation has been studied extensively in
both species. In the case of A. suum, the sperm extends a Membrane Microdomains crescent-shaped ！at pseudopod with distinguishable MSP In mammalian spermatozoa, capacitation (the collective ，ber bundles (Sepsenwol et al., 1989). Spermatids can be physiological processes that render sperm competent for activated in vitro using either S. griseus proteases (25 mg/ml)
fertilization) accompanies the remodeling of membrane or a vas deferens extract (VDX) from A. suum (Sepsenwol
microdomains that are responsible for the acrosome reac- and Taft, 1990). Sperm activated using proteases extend a
tion, sperm–egg recognition, and the ultimate fusion of the pseudopod, but are unable to crawl, while activation with sperm and egg plasma membranes (Gadella et al., 2008; VDX induces crawling at an average instant velocity of
Nixon and Aitken, 2009). In other cell types, these choles- 30.3 16.2 mm/min (Royal et al., 1997). In contrast, sper-
terol-enriched dynamic microdomains, often referred to as matozoa from C. elegans possess a less-！at pseudopod that
lipid rafts, are proposed to functionally cluster membrane lacks visible MSP ，ber bundles. Spermatids are activated in
proteins within common intracellular pathways (Simons and vitro using either S. griseus proteases (200 mg/ml), Trietha-
Toomre, 2000; Golub et al., 2004). Recently, this view of nolamine (TEA), a weak base that promotes an increase in
‘‘patchy’’ and regionalized membranes has been discussed intracellular pH (Ward et al., 1983), or the cationic ionophore (Engelman, 2005). Monensin (Nelson and Ward, 1980). C. elegans spermato-
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MOTILITY IN NEMATODE SPERM
Polymerization Organizing Protein (MPOP) recruits cytosol- In amoeboid sperm, the plasma membrane is a very
dynamic structure that must maintain the integrity of inter- ic proteins to the membrane surface (LeClaire et al., 2003). actions between proteins in common signaling pathways. Elongation and bundling of the ，laments into ，bers occurs at
Thus, we speculate that the integrity of protein interactions is the leading edge of the pseudopod, forming a complex maintained due to the presence of membrane microdomains intertwined network of MSP (Roberts and Stewart, 2000; as compelling evidence supports this hypothesis for C. Bottino et al., 2002). This cytoskeletal network is connected elegans sperm (see Fig. 2). For instance, the SPE-8 group mechanically to the substratum through the membrane, of proteins (that include SPE-8, SPE-12, SPE-19, SPE-27, allowing directional movement of the cell. In C. elegans and SPE-29), responsible as a trigger for spike formation sperm, cells crawl by traction exerted through plasma mem- and further pseudopod extension, is thought to function as a brane proteins on the substrate. This attachment can be multicomponent complex that involves the interaction of inhibited by antibodies directed against the membrane pro- membrane and cytoplasmic signaling proteins (Geldziler et teins TR11 and SP56, but not by the anti MSP antibody, al., 2005; see Fig. 2A). Other membrane microdomains TR20 (Pavalko et al., 1988). Altogether, these results lead to could be hypothesized in the docking and fusion of MOs to the hypothesis that MSP is localized to the cytoplasmic the plasma membrane (see Fig. 2B) as FER-1, a protein lea！et of the plasma membrane and other proteins involved involved in vesicle fusion, has a human homolog that inter- in attachment and traction are localized to the extracellular acts with Caveolin-3 (Matsuda et al., 2001), a membrane lea！et. If these proteins are co-localized in discrete regions microdomain marker. FER-1 is hypothesized to promote of attachment we can speculate that crawling in nematode vesicle fusion through protein–protein interactions with sperm might be similar to the formation of focal adhesions in SNARE proteins (Washington and Ward, 2006), a complex cells with an actin-based motility where ordered membrane localized in cholesterol-enriched microdomains (Lang, microdomains play an important role (Wozniak et al., 2004; 2007). Gaus et al., 2006; see Fig. 2C).
A supporting argument on the presence of membrane Collectively, this body of information leads us to hypoth- microdomains is revealed by cholesterol localization in esize that membrane microdomains are present in nema- spermatids and the protein redistribution that occurs during tode sperm and involved in the processes of sperm pseudopod extension. Matyash et al. (2001) used a choles- activation, MO fusion to the plasma membrane, and motility terol ！uorescent analog, dehydroergosterol, to localize its acquisition, rendering the cell competent for successful distribution in C. elegans during development, showing that fertilization. Thus, future experiments in nematode sperm males displayed a strong labeling in dispersed cytoplasmic motility can be designed to test whether proteins that are structures of the spermatid. Also in spermatids, proteins that redistributed in the plasma membrane may be released or are important for fertilization change their localization sub- sequestered within different microdomains and localize to sequent to the extension of the pseudopod (Singson et al., speci，c morphological regions (in an analogous fashion with 2008). For instance, SPE-9 is an EGF repeat transmem- mammalian spermatozoa) such as the pseudopod, where brane protein localized homogeneously over the spermatid they can move from the tip to the base, thereby recycling plasma membrane, while after pseudopod extension it is receptors for sperm–egg fusion and conferring attachment found exclusively on the pseudopod (Zannoni et al., 2003). of the cell to the substrate resulting in successful fertilization. Other examples of this reorganization include the proteins
SPE-38, a four-pass integral membrane protein (Chatterjee
et al., 2005), a TRPC calcium channel known as TRP-3 or ION PHYSIOLOGY AND SIGNAL TRANSDUCTION SPE-41 (Xu and Sternberg Paul, 2003), and the MO marker DURING PSEUDOPOD EXTENSION 1CB4 (Okamoto and Thomson, 1985). All of these are In ！agellated spermatozoa, membrane rearrangements localized to MOs prior to pseudopod extension and change
are hypothesized to have an effect on the biophysical prop- their distribution upon fusion to the plasma membrane. SPE-
erties of the plasma membrane with an impact on ion 38 co-localizes to the pseudopod with SPE-9, TRP-3 redis-
channel and/or enzymatic activity on the cell surface tributes to the plasma membrane in both the cell body and (Kopf et al., 1999). These changes in ionic ！uxes are the pseudopod, and the 1CB4 marker is maintained in the
required for an increase in sperm motility and to achieve MOs bodies (see Fig. 2B).
capacitation through plasma membrane hyperpolarization, In Ascaris sperm, there is also evidence for membrane 2alkalization of intracellular pH, increased intracellular Caþ microdomains. MSP nucleation and elongation occurs concentrations, and the initiation of intracellular signaling at the leading edge of the pseudopod where the MSP
Figure 2. Hypothetical involvement of membrane microdomains during pseudopod extension. Membrane microdomains (lipid rafts) are
sphingolipid- and cholesterol-enriched signaling platforms involved in the spatial and temporal regulation of processes triggered at the cell surface. A: The SPE-8 group of proteins (involved in pseudopod extension) is an agglomerate of membrane and cytosolic proteins present in a multicomponent complex that affect a signaling response upon an extracellular cue at the cell surface. B: The membraneous organelles are candidate sites for membrane microdomains. The co-localization of putative cholesterol-enriched regions and proteins involved in sperm–egg interaction (that change distribution upon fusion of membranous organelles to the plasma membrane) supports the model for protein release and recruitment of microdomain-based rearrangements. C: The crawling of nematode spermatozoa involves discrete membrane regions of ，lament nucleation and substrate attachment that translocate proteins from the tip to the base of the pseudopod, this protein ‘‘recycling’’ could also involve molecules implicated in sperm–egg fusion.
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Figure 3. Ionic conductance plays an important role in the acquisition of motility in amoeboid sperm. Although studied to a lesser extent, ion ！uxes are necessary for pseudopod extension and motility maintenance. The blockage of Clchannels induces sperm activation due to changes in irþþthe resting membrane potential and/or the accumulation (or transport) of HCOinto the cytoplasm, causing alkalization. Naand Kions are 3þþnecessary for sperm activation and could also affect membrane potential. An alternative is that the exchange of Naand H(in a manner similar to the in vitro activator Monensin) can lead to an increase of intracellular pH, affecting properties of the plasma membrane. Cytoplasmic alkalization initiates the establishment of a pH gradient that maintains motility in the pseudopod through MSP elongation and protein phosphorylation. Another upstream effector of protein phosphorylation is the SPE-8 group of proteins that initiate pseudopod extension and the calcium-dependent machinery that induces membranous organelle fusion to the plasma membrane and putatively regulates dynamics of the MSP cytoskeleton. The study of ion physiology in nematode sperm will provide an integrative understanding of the dynamic processes that lead to acquisition of sperm motility and fertilization in different taxa.
cascades that involve protein phosphorylation events (for as Monensin and Valinomycin has provided insight on reviews see Darszon et al., 2008; Suarez, 2008; Abou-haila changes in sperm intracellular pH necessary to maintain and Tulsiani, 2009). pseudopod extension in both C. elegans and A. suum In nematode sperm, ionic ！uxes during pseudopod ex- (Nelson and Ward, 1980; Roberts and King, 1991). Howev- tension have been studied to a lesser extent (Fig. 3). Ma- er, this cytosolic alkalization is coupled to the exchange of þþchaca et al. (1996) used the patch clamp technique to protons with Na, in the case of Monensin, and K, in the
investigate voltage-sensitive ion channel activities in C. case of Valinomycin, making it unclear whether spermatid elegans sperm and calculated the spermatid’s resting activation is coupled to cytosolic alkalization in general or to þþspeci，c changes in Naor Kions. In this regard, it has been potential (V? 8.07 2.26 mV) in Nystatin-perforated Rwhole-cell experiments. These investigators discovered shown that both ions are necessary for in vitro pseudopod several voltage-sensitive ion channel activities in both sper- extension as triethanolamine and Monensin spermatid acti- þþmatocytes and residual bodies during spermatogenesis, vation are reduced or abolished when Naand Kare
replaced in the medium (Nelson and Ward, 1980; Ward et although only an inward-rectifying chloride channel (Cl) irþwas detected in spermatids upon hyperpolarization. In the al., 1983). Activation with Pronase is also reduced when K
same study, the chloride channel inhibitor 4,40-di-isocyana- ions are replaced, although this activator does not alter 0tostilbene-2,2-disulfonic acid was found to induce activation intracellular pH (Ward et al., 1983). Nelson and Ward
(1980) also suggested a trigger for pseudopod extension in spermatids, suggesting an important role of Clchannels irþdue to changes in membrane potential based on K-depen- in the process of pseudopod extension. Sperm activation by
dent mechanisms (Nelson and Ward, 1980). To our knowl- the blockage of Clis suggested to act by either a change in iredge, the mechanisms for how nematode sperm membrane membrane potential or by a transport of HCOions into the 3potential is affected during pseudopod extension using any cytoplasm, resulting in an increase of intracellular pH
in vitro activator have not been investigated to date. (Machaca et al., 1996). Indeed, the use of ionophores such
Mol Reprod Dev 77:739–750 (2010) 744
OTILITY IN NEMATODE SPERM M 2ogy and movement recovered normally. The Caþ channel Intracellular pH
blocker nicardipine was reported in the same article to have As is the case in ！agellated sperm, an increase in intra-
a similar effect. These investigators also observed that cellular pH is necessary for motility acquisition in nematode
spermatids from the spe-8 and spe-12 mutants arrest in the sperm. This was ，rst suggested when spermatids treated
‘‘spike’’ stage after activation with Pronase. This spike arrest with Monensin showed a pH dependence on pseudopod
could be overcome and pseudopod extension occurred extension (Nelson and Ward, 1980). Weak bases, including following treatment with TFP, and the other Calmodulin TEA, also exerted pseudopod extension accompanied by an inhibitors, although TEA and Monensin had the same effect. increase in intracellular pH (Ward et al., 1983). In this study, All of these observations were reported to occur in the the authors reported that cytoplasmic alkalization is suf，- 2absence of external Caþ, suggesting the involvement of cient to trigger spermatid activation and that removal of TEA 2internal Caþ stores. Furthermore, it was shown that motile did not affect pseudopod morphology. However, in A. suum
spermatozoa treated with the Calmodulin inhibitors stop spermatozoa, treatment with weak acids caused loss of
motility and rounding up of the pseudopod. Shakes and motility, disassembly of the MSP cytoskeleton and, conse-
Ward (1989) were not able to detect Calmodulin in sperma- quently, compromised pseudopod integrity (Roberts and tids using antibodies that cross-react with nematode Cal- King, 1991). Thus, maintenance of a speci，c intracellular modulin. Together, this evidence complemented with the pH, putatively buffered by HCO, is crucial for pseudopod 3known effects of TFP, CPZ, and W7 on processes unrelated integrity and crawling of spermatozoa. Further, the MSP to Calmodulin, led the authors to conclude as unlikely the cytoskeleton polymerization involved in amoeboid move- effect of these drugs on the inhibition of Calmodulin, as this ment (protrusion and retraction) is regulated by an intracel-
would suggest a rather odd role of blocking pseudopod lular pH gradient along the pseudopod (King et al., 1994; 2extension by this Caþ-binding protein, given that Calmod- Italiano et al., 1999). Using the pH-sensitive ！uorescent ulin in ！agellated sperm plays an important role in hyper- indicator BCECF, King et al. (1994), demonstrated an intra- activation of motility, capacitation, and acrosome reaction cellular alkalization of 0.25 pH units during activation of
through protein tyrosine phosphorylation events. spermatids using vas deferens extract (VDX). The weak 2Subsequently, the Caþ-binding protein, Calreticulin bases, TEA and NHCl, induced a similar pH increase of 4(CRT-1), was observed to be present in the cytoplasm of 0.21 and 0.32 units respectively, although, only blebs or C. elegans sperm. The crt-1 mutant spermatozoa appeared ‘‘spikes’’ were formed by the spermatid and no pseudopod to have slightly shorter pseudopods and nuclei that were off was extended. In this study, they also showed an increase of center, suggesting a role for CRT-1 in the late stages of 0.15 pH units between the tip of the pseudopod where spermatogenesis (Park et al., 2001). The same research alkalization correlates with MSP ，bers assembly, and the 2group discovered the presence of Calcineurin, a Caþ/Cal- base of the pseudopod where acidi，cation promotes disas- modulin-dependent serine/threonine protein phosphatase sembly of the MSP cytoskeleton. Thus, mechanisms of (PP2B) in C. elelgans sperm, with cnb-1 mutants sharing intracellular pH regulation are involved in the trigger and short pseudo- the same phenotype as crt-1 mutant sperm –maintenance of motility forces in amoeboid sperm (Bottino et pod and reduced size (Bandyopadhyay et al., 2002). Wheth- al., 2002).
er, Calmodulin, or a Calmodulin-dependant mechanism, 2regulates Caþ dynamics during pseudopod extension is 22Intracellular Caþ still unclear, although Caþ-regulated machinery is certainly 22Caþ is a key second messenger that regulates sperm present in C. elegans spermatids. This Caþ-dependent
capacitation and motility in ！agellated sperm together with machinery is complemented by FER-1, a protein of the ferlin 22Caþ-binding proteins (Abou-haila and Tulsiani, 2009). In family involved in the Caþ-mediated MO fusion to the 2nematode sperm, the role of Caþ during motility acquisition plasma membrane (Washington and Ward, 2006). FER-1 has only been studied in C. elegans; however, these studies is characterized by multiple C2 domains that function as 22yield insights not only on the physiological stages of Caþ Caþ sensors and interact with phospholipids and proteins of signaling during pseudopod extension but also on the con- the membrane fusion machinery during exocytosis (Bai and served role of this ion during exocytosis and fertilization. In Chapman, 2004). It has also been demonstrated that intra- 21980, Nelson and Ward demonstrated that pseudopod ex- cellular Caþ stores are involved in the proper fusion of MOs 2tension is not induced with either of the Caþ ionophores to the plasma membrane since the membrane-permeable 22A23187 or X537A. External Caþ is not necessary either for Caþ chelator BAPTA-AM prevented MO fusion in a con- the trigger of spermatid activation with any of the in vitro centration-dependent manner (Washington and Ward, activators, Monensin, TEA or Pronase, since removal of this 2006). In Drosophila melanogaster, the ferlin gene mis，re
ion from the medium has little or no effect on pseudopod (mfr) is a homolog of C. elegans fer-1, and is involved in early extension (Nelson and Ward, 1980; Ward et al., 1983). embryogenesis and egg patterning in females, and in the Intriguing results from Shakes and Ward in 1989 showed completion of fertilization in males (Ohsako et al., 2003; that the Calmodulin inhibitors Tri！uoperazine (TFP), Chlor- Smith and Wakimoto, 2007). Successful fertilization in D. promazine (CPZ), and Naphtalenesulfonamide (W7) can melanogaster involves sperm entry (by puncturing a hole in induce pseudopod extension, although the pseudopod is the egg oolemma without the involvement of membrane abnormal in that it lacks villar projections and is devoid of fusion) followed by sperm plasma membrane break down membrane movement (Shakes and Ward, 1989). Once the (PMBD), nuclear decondensation, and the formation of a Calmodulin inhibitor was removed, the pseudopod morphol- male pronucleus. A testis isoform of Mfr containing ，ve C2
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domains and a transmembrane domain is required for sperm domain-containing nonreceptor tyrosine kinase with an un- 2PMBD, a process also mediated by Caþ-dependent mem- known substrate (WormBase, release WS203). SPE-6, a brane interactions between the acrosome and the plasma homolog of serine/threonine casein kinase 1, appears to be membrane (Smith and Wakimoto, 2007). an inhibitory kinase as spermatid activation requires that 2The ，nal piece of evidence for Caþ dynamics during SPE-6 is repressed by upstream components such as SPE- pseudopod extension involves a Transient Receptor Poten- 8 (Muhlrad and Ward, 2002). Conversely, SPE-19 is a novel tial Channel (TRPC) homolog, TRP-3, present in C. elegans protein with a predicted single internal transmembrane do- spermatozoa and required for sperm–egg interaction during main and a large number of serine/threonine phosphoryla- fertilization (Xu and Sternberg Paul, 2003). As explained tion sites on the intracellular portion, putatively involved in previously, TRP-3 is localized in the MO in spermatids and, the reception/transduction of activator signals (Geldziler et upon pseudopod extension, the channel is translocated to al., 2005). However, changes in protein phosphorylation the cell body due to its delivery from MOs fusion to the during pseudopod extension have never been examined in 2plasma membrane. Using the ！uorescent Caþ indicator C. elegans.
Fura-2, the authors investigated the mechanistic defect of Important evidence of regulatory kinase cascades comes 2trp-3 sperm on calcium signaling. In Caþ-free medium, two from studies in A. suum. The identi，cation of molecular 2types of Caþ in！uxes were observed in mature spermato- components that regulate polymerization of MSP leading zoa. One in！ux appeared to be the result of constitutively to the acqusition of sperm motility has been facilitated by the 2active Caþ-permeable channels (CAC) that open immedi- in vitro reconstitution of ，ber assembly (Italiano et al., 1996). 2ately upon introduction of extracellular Caþ. Intracellular These ，bers were shown to elongate due to the activity of a 2Caþ stores regulated the second in！ux since treatment with cytoplasmic soluble factor and an integral vesicle protein 2ionomycin and thapsigargin induced a large Caþ in！ux after generating the protrusive force necessary for psedupod introduction of this ion in the medium. The authors described extension (Roberts et al., 1998). Thus, accessory proteins 2the latter as a Store Operated Caþ Entry (SOCE) channel. responsible for MSP ，ber elongations were identi，ed in the 2However, Caþ release from intracellular stores was not cytoplasmic fraction and in membrane vesicles of A. suum detected during treatment with either ionomycin or thapsi- spermatozoa. The ，rst evidence for protein phosphorylation gargin. The SOCE response was reduced in trp-3 mutant in MSP polymerization was that the in vitro reconstitution spermatozoa, suggesting TRP-3 is necessary for SOCE assay required ATP to elongate ，bers (Italiano et al., 1996). activity and successful fertilization of the oocyte. In contrast, Later work showed that the protein tyrosine phosphatase spermatids show minimal CAC activity and much lower YOP, from Yersenia enterocolitica, blocked in vitro ，ber
SOCE. Transient Receptor Potential channel homologs are assembly (Miao et al., 2003), leading to the identi，cation of
also present in mammalian spermatozoa (Trevi~no et al., the MSP Polymerization Organizing Protein (MPOP), an 2001), and are thought to play a role in motility regulation integral membrane phosphoprotein localized at the leading (Castellano et al., 2003) and the acrosome reaction edge of the sperm lamellipod where its tyrosine phosphory- (Jungnickel et al., 2001; De Blas et al., 2009). lated state can be modulated by intracellular pH (LeClaire et Summarizing this evidence, we hypothesize that extra- al., 2003). Yi et al. (2007) identi，ed a serine/threonine 2cellular Caþ is not necessary to trigger spermatid activa- Casein kinase 1, MSP polymerization-activating Kinase tion, although a release from intracellular stores is (MPAK), that is recruited by MPOP to the plasma mem- 2necessary for MO fusion and pseudopod extension. A Caþ brane, regulating MSP polymerization by phosphorylation of signaling machinery is present in C. elegans sperm that the MSP Fiber Protein 2 (MFP2), a protein that can bind MSP 2involves Caþ-binding proteins such as Calreticulin and ，laments and accelerate their elongation (Buttery et al., Calcineurin, a Calmodulin-dependent protein phosphatase. 2003; Grant et al., 2005). Curiously, MPAK shares homology 2Thus, Caþ ！uctuations regulated by Calmodulin dependent with the Casein kinase 1 SPE-6 from C. elegans (Yi et al., mechanisms and Store Operated Calcium channels should 2007). MPAK, MFP2, and a tyrosine-phosphorylated protein be investigated in further detail in nematode sperm to es- (putatively MPOP) are also involved in the extension of 2tablish relationships to Caþ-dependent signaling cascades spikes or blebs during sperm activation and in the in vitro that occur in ！agellated spermatozoa. reconstitution of ，lopodia in A. suum (Rodriguez et al., 2005;
Miao et al., 2007).
In summary, it is proposed that the activation of the Protein Phosphorylation spermatid, extension of the pseudopod, and maintenance Protein phosphorylation cascades are events that or- of motility are regulated by phosphorylation cascades in A. chestrate the acquisition of motility in both ！agellated and suum and the same might hold true for C. elegans. Both nematode sperm. There is evidence from both C. elegans nematode spermatozoa possess a Casein kinase 1 that and A. suum that these events regulate the extension of a regulates the initiation of pseudopod extension and, puta- pseudopod. In the case of C. elegans, genes identi，ed in tively, the nucleation and elongation of MSP ，bers during microarray studies as spermatogenesis-enriched include a acquisition of motility. MFP3, a protein involved in sperm surprisingly large number of kinases and phosphatases retraction (see below), also shows homology with the un- (Reinke et al., 2000). The SPE-8 group of proteins, respon- characterized sperm-enriched proteins SSQ1-4 from C. sible for triggering pseudopod extension, includes members elegans (Yi et al., 2009). Whether other proteins from this that are thought to be involved in protein phosphorylation as machinery are conserved between nematode sperm is a concluded by gene sequencing data. SPE-8 is an SH2- question that remains to be answered.
746 Mol Reprod Dev 77:739–750 (2010)
OTILITY IN NEMATODE SPERM M
activation overcomes an increase in intracellular pH and is Finally, a number of recent reports have identi，ed addi-
tional signaling proteins that regulate both mammalian and not affected by anaerobic metabolism (Ward et al., 1983). nematode sperm motility. For example, in A. suum, the Although this presumed endogenous protease has yet to be serine/threonine protein phosphatase 2A (PP2A) regulates identi，ed, the proposed model ，ts with the recently reported the cytoskeletal dynamics of sperm motility by dephosphor- novel function of SPE-4, a member of the activation inhibi- ylating MFP3, an event that results in the disassembly of tory pathway and a homolog of the human presenilin gene MSP ，laments and cell body retraction (Yi et al., 2009). In PS1, that encodes a protein with predicted proteolytic activi- mouse sperm, the Calyculin A-sensitive protein phospha- ty targeting the transmembrane domains of integral mem- 2tases, PP1A and PP2A, regulate phosphorylation events of brane proteins and is also known to perturb Caþ
！agellar proteins involved in hyperactivation of motility (Goto homoestasis (Gosney et al., 2008). These investigators and Harayama, 2009). These protein phosphatases are also propose a model in which FER-1 is a target of SPE-4 2thought to suppress full activation of protein kinase A (PKA) proteolytic activity, as both are present in MOs and a Caþ
in mammalian sperm. It has also been suggested that increase is necessary for fusion of these organelles with the activation of PKA promotes activity of phosphatidylinositol 3 plasma membrane. Thus, under this hypothesis, we can -kinase (PI3-K), by downregulation of protein kinase C assume a role for anaerobic metabolism and intracellular pH (PKC) activity (Breitbart et al., 2006). In C. elegans sperm, in the activation of SPE-4, which ultimately leads to the 2the crosstalk between a putative PI3-K and CIL-5, a PI 5- fusion of MOs based on intracellular Caþ stores and the
phosphatase, is thought to regulate pseudopod extension by initiation of pseudopod extension and acquisition of motility controlling levels of phosphatidylinositol-3,4,5-triphosphate under the control of aerobic metabolism.
(PI(3,4,5)P3), since the PI3-K antagonist, Wortmannin acts To date, the in vivo extracellular initiation signals that lead as an in vitro sperm activator, promoting pseudopod exten- to spermatid activation in nematodes are currently unknown. sion (Bae et al., 2009). However, Abou-haila and Tulsiani Sepsenwol and Taft (1990) suggested that according to their (2009) discussed differential effects of Wortmannin on mam- unpublished results, the vas deferens factor responsible for malian sperm capacitation, concluding that the role of PI3-K spermatid activation in A. suum is a pepsin-sensitive glyco- in ！agellated sperm remains elusive. Thus, it is clear that protein with a molecular mass of approximately 60 kDa. further investigations in protein phosphorylation and phos- They further argued that their model is congruent with the phoinositide signaling cascades, in both mammalian and inhibition of VDX activation by the serine-protease inhibitor nematode sperm, will shed light on the role of conserved PMSF, although serine proteases fail to initiate pseudopod fertilization processes among different taxa. extension in A. suum. In C. elegans, spermatids can be
activated by Pronase, trypsin, and chymotrypsin (Ward et
al., 1983) and a recent paper has shown that the serine
protease inhibitor SWM-1, which contains two trypsin-like NEMATODE SPERM METABOLISM, PROTEASES inhibitor domains, is responsible for preventing pseudopod AND THE CONNECTION BETWEEN IN VITRO AND IN extension in male seminal ！uid (Stan，eld and Villenueve, VIVO ACTIVATION OF SPERMATIDS 2006). Currently, all evidence suggests a role for proteases How changes in cellular metabolism regulate the exten- triggering sperm activation, although the connection be- sion and maintenance of nematode sperm pseudopods is tween this process and the downstream physiological re- poorly understood. While some nematode spermatozoa sponses that accompany pseudopod extension remain such as Nipposfrongyfus (Wright and Sommerville, 1984), elusive.
and Nematospiroides (Wright and Sommerville, 1985) acti-
vate and crawl under aerobic conditions, others including
spermatozoa from A. suum appear to be obligate anaerobes CONCLUSION that require high pCOfor activation and crawling 2The physiological activation of cell motility is a response (Sepsenwol and Taft, 1990). In motile C. elegans sperma-
tozoa, sodium azide and other mitochondrial poisons such triggered from cues in the environment. Spermatozoa from as oligomycin and dinitrophenol prevent spermatid activa- the deuterostomate lineage initiate motility and become tion and arrest pseudopod movement, suggesting that mi- competent for fertilization due to environmental cues that tochondrial aerobic metabolism is necessary for pseudopod affect intracellular processes such as membrane remodel- extension and motility (Roberts and Ward, 1982b; Ward et ing, cytosolic alkalization, increased ionic in！ux, and in-
al., 1983). Interestingly, 2-deoxyglucose prevents pseudo- creased metabolism, resulting in the modulation of pod extension in spermatids activated with TEA while pro- ！agellar beat parameters, acrosomal exocytosis, and fertili- nase-activated spermatozoa were insensitive to this zation (Darszon et al., 2008; Suarez, 2008; Abou-haila and treatment. The observation that 2-deoxyglucose inhibition Tulsiani, 2009). Studies on the nematodes C. elegans andA. could be counteracted by the addition of fructose led the suum have revealed that these physiological responses are authors to propose a model in which an increase in intracel- also true for the acquisition of motility in amoeboid sperma- lular pH triggers the release of a protease that acts on a tozoa, although the mode of locomotion is a pseudopod, surface protein leading to membrane rearrangements, and rather than a ！agellum. Recapitulating the steps that lead to ultimately the initiation of aerobic metabolism. In assuming pseudopod extension and acquisition of motility in nematode that the release of an endogenous sperm protease requires sperm, we propose that macromolecular rearrangements ATP from glycolysis, this model explains why Pronase and increased ！uidity of the plasma membrane must have
Mol Reprod Dev 77:739–750 (2010) 747
Molecular Reproduction & Development RAIRE-ZAMORA AND CARDULLO F
consequences for the biophysical properties of the mem- Buttery SM, Ekman GC, Seavy M, Stewart M, Roberts TM. 2003. brane. Thus, cell surface protein redistribution in microdo- Dissection of the Ascaris sperm motility machinery identi，es key
mains might be responsible for the maintenance of quality, proteins involved in major sperm protein-based amoeboid loco- duration, and strength of signaling cascades during pseu- motion. Mol Biol Cell 14:5082–5088.
dopod extension as proposed in other cell types (Golub et Castellano LE, Trevi~no CL, Rodrıguez D, Serrano CJ, Pacheco J, al., 2004). These rearrangements can be triggered by Tsutsumi V, Felix R, Darszon A. 2003. Transient receptor poten- þþchanges in ionic ！uxes involving Na, K, and Cl ions, tial (TRPC) channels in human sperm: Expression, cellular having a putative effect on membrane potential, and leading localization and involvement in the regulation of ！agellar motility. 2to the activation of a Caþ-dependent machinery necessary FEBS Lett 541:69–74.
for MOs fusion and pseudopod extension. The maintenance Chatterjee I, Richmond A, Putiri E, Shakes D, Singson A. 2005. The of a pseudopod that confers motility to the sperm is achieved Caenorhabditis elegans spe-38 gene encodes a novel four-pass by an exquisite regulation and establishment of a pH gradi- integral membrane protein required for sperm function at fertili- ent along the cell, coupled to protein phosphorylation in zation. Development 132:2795–2808.
signaling cascades that orchestrate cytoskeletal dynamics. Chu DS, Liu H, Nix P, Wu TF, Ralston EJ, Yates JR III, Meyers BJ. Altogether, these processes must have as an outcome 2006. Sperm chromatin proteomics identi，es evolutionary con-
the successful fertilization of an egg by a highly motile served fertility factors. Nature 443:101–105.
spermatozoon. Despite morphological differences, nema- Darszon A, Guerrero A, Galindo BE, Nishigaki T, Wood CD. tode sperm have recently been considered as a good model 2008. Sperm-activating peptides in teh regulation of ion for the development of human male contraceptives, due to ！uxes, signal transduction and motility. Int J Dev Biol 52: the identi，cation of sperm chromatin fertility factors that are 595–606.
evolutionarily conserved among mice, humans, and nema- De Blas GA, Darszon A, Ocampo AY, Serrano CJ, Castellano LE, todes (Chu et al., 2006). In this review, we propose that Hernandez-Gonzalez EO, Chirinos M, Larrea F, Beltran C, similar arguments may be made when comparing the com- Trevi~no CL. 2009. TRPM8, a versatile channel in human sperm. mon physiological factors that lead to acquisition of motility in PLoS ONE 4:e6095.
these systems. We strongly believe that our hypotheses can Engelman DM. 2005. Membranes are more mosaic than ！uid.
be tested using an integrative approach—one that combines Nature 438:578–580.
proteomics, biochemistry, genetic, and imaging techniques, Gadella BM, Tsai PS, Boerke A, Brewis IA. 2008. Sperm head leading to a better understanding of the evolutionary con- membrane reorganisation during capacitation. Int J Dev Biol served processes that lead to the acquisition of sperm 52:473–480.
motility and fertilization not only among the phylum Nema- Gaus K, Le Lay S, Balasubramanian N, Schwartz MA. 2006. toda but across all eumetazoans. Integrin-mediated adhesion regulates membrane order. J Cell
Geldziler B, Chatterjee I, Singson A. 2005. The gentic and molecu-
lar analysis of spe-19, a gene required for sperm activation in ACKNOWLEDGMENTS Caenorhabditis elegans. Dev Biol 283:424–436. We would like to thank the anonymous reviewers for their Golub T, Wacha S, Caroni P. 2004. Spatial and temporal control of valuable comments that considerably improved this manuscript. signaling through lipid rafts. Curr Opin Neurobiol 14:542–550. J.J.F.Z. would like to dedicate this work to the memory of Dr. Marco Gosney R, Liau W-S, LaMunyon CW. 2008. A novel function for the T. Gonzalez-Martınez. presenilin family member spe-4: Inhibition of spermatid activation in Caenorhabditis elegans. BMC Dev Biol 8:1–14. Goto N, Harayama H. 2009. Calyculin A-sensitive protein REFERENCES phosphatases are involved in maintenance of progressive Abou-haila A, Tulsiani DRP. 2009. Signal transduction pathways movement in mouse spermatozoa in vitro by suppression of that regulate sperm capacitation and the acrosome reaction. autophosphorylation of Protein Kinase A. J Reprod Dev Arch Biochem Biophys 485:72–81. 55:327–334.
Bae Y-K, Kim E, L’Hernault SW, Barr MM. 2009. The CIL-1 PI 5- Grant RP, Buttery SM, Ekman GC, Roberts TM, Stewart M. 2005. phosphatase localizes TRP polycystins to cilia and activates Structure of MFP2 and its function in enhancing MSP polymeri- sperm in C. elegans. Curr Biol 19:1–9. zation in Ascaris sperm amoeboid motility. J Mol Biol 347: Bai J, Chapman ER. 2004. The C2 domains of synaptotagmin- 583–595.
partners in exocytosis. Trends Biochem Sci 29:143–151. Italiano JE, Roberts TM, Stewart M, Fontana C. 1996. Reconstitu- Bandyopadhyay J, Lee J, Lee J, Lee JI, Yu J-R, Jee C, Cho J-H, tion in vitro of the motile apparatus from the amoeboid sperm of Jung S, Lee MH, Zannoni S, Singson A, Kim DH, Koo HS, Ahnn J. Ascaris shows that ，lament assembly and bundling move mem- 2002. Calcineurin, a calcium/calmodulin-dependent protein branes. Cell 84:105–114.
phosphatase, is involved in movement, fertility, egg laying, and Italiano JE, Stewart M, Roberts TM. 1999. Localized depolymar- growth in Caenorhabditis elegans. Mol Biol Cell 13:3281–3293. ization of the major sperm protein cytoskeleton correlates with Bottino D, Mogilner A, Roberts T, Stewart M, Oster G. 2002. How the forward movement of the cell body in the amoeboid move- nematode sperm crawl. J Cell Sci 115:367–384. ment of nematode sperm. J Cell Biol 146:1087–1096.
Breitbart H, Rubinstein S, Etkovitz N. 2006. Sperm capacitation is Jungnickel MK, Marrero H, Birnbaumer L, Lemos JR, Flormann regulated by the crosstalk between protein kinase A and C. Mol HM. 2001. Trp2 regulates entry of Ca2þ into mouse sperm Cell Endocrinol 252:247–249. triggered by egg ZP3. Nat Cell Biol 3:499–502.
Mol Reprod Dev 77:739–750 (2010) 748