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The physiological acquisition of amoeboid motility in nematode sperm: Is the tail the only thing the sperm lost?

2010; Wiley; Volume: 77; Issue: 9 Linguagem: Inglês

10.1002/mrd.21193

1098-2795

Juan J Fraire-Zamora, Richard A. Cardullo,

Protist diversity and phylogeny

Molecular Reproduction and DevelopmentVolume 77, Issue 9 p. 739-750 Review ArticleFree Access The physiological acquisition of amoeboid motility in nematode sperm: Is the tail the only thing the sperm lost? Juan J. Fraire-Zamora, Juan J. Fraire-Zamora Department of Biology and the Graduate Program in Evolution, Ecology, and Organismal Biology, University of California, Riverside, CaliforniaSearch for more papers by this authorRichard A. Cardullo, Corresponding Author Richard A. Cardullo cardullo@ucr.edu Department of Biology and the Graduate Program in Evolution, Ecology, and Organismal Biology, University of California, Riverside, CaliforniaDepartment of Biology, University of California, Riverside, CA 92521.Search for more papers by this author Juan J. Fraire-Zamora, Juan J. Fraire-Zamora Department of Biology and the Graduate Program in Evolution, Ecology, and Organismal Biology, University of California, Riverside, CaliforniaSearch for more papers by this authorRichard A. Cardullo, Corresponding Author Richard A. Cardullo cardullo@ucr.edu Department of Biology and the Graduate Program in Evolution, Ecology, and Organismal Biology, University of California, Riverside, CaliforniaDepartment of Biology, University of California, Riverside, CA 92521.Search for more papers by this author First published: 23 August 2010 https://doi.org/10.1002/mrd.21193Citations: 8AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Nematode spermatozoa are highly specialized amoeboid cells that must acquire motility through the extension of a single pseudopod. Despite morphological and molecular differences with flagellated spermatozoa (including a non-actin-based cytoskeleton), nematode sperm must also respond to cues present in the female reproductive tract that render them motile, thereby allowing them to locate and fertilize the egg. The factors that trigger pseudopod extension in vivo are unknown, although current models suggest the activation through proteases acting on the sperm surface 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 flagellated sperm) that involve membrane rearrangements in response to extracellular cues that initiate various signal transduction pathways. An integrative approach to the study of nonflagellated spermatozoa will shed light on the identification of unique and conserved processes during fertilization among different taxa. Mol. Reprod. Dev. 77: 739–750, 2010. © 2010 Wiley-Liss, Inc. Abbreviations: MSP, major sperm protein; MO, membranous organelle; MPOP, MSP polymerization; organizing protein; SOCE, store operated calcium entry; TEA, triethanolamine; VDX, vas deferens extract. INTRODUCTION Fertilization is common to all multicellular organisms that reproduce sexually. In this process, two gametes, typically haploid cells, fuse to form a new individual. In order for successful fertilization to occur, two events are necessary. First, the gametes must find one another so that they come into direct physical contact. Second, upon sperm–egg binding, a complex series of biochemical and physiological events must occur, culminating in fusion and egg activation (Yanagimachi, 1994). The “typical” fertilization process involves a sessile egg that is encountered by a highly motile spermatozoon. Most frequently, the spermatozoon is propelled by a microtubule-based flagellum that brings the gametes into sufficiently close contact to ultimately result in fertilization. Although this general view of fertilization is often assumed to be universal, careful reviews reveal that many taxa produce nonflagellated sperm (Morrow, 2004; see Fig. 1A). Figure 1Open in figure viewerPowerPoint Not everyone needs a tail to get around. Species with nonflagellated 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, nonflagellated sperm are not uncommon. The phylogenetic tree shows Phyla that contain at least one species bearing nonflagellated spermatozoa (in red); Phyla containing only nonflagellated spermatozoa (in blue); and Phyla containing only flagellated spermatozoa (in black) (Phylogenetic tree modified 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 flagellum 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 fillopodia-like projections (spikes), and the confinement of organelles into the cell body, while a pH gradient is formed from the tip to the base of the pseudopod. Perhaps, the most studied taxonomic group with motile nonflagellated sperm is the Phylum Nematoda. This phylum is extremely diverse with both free-living and parasitic representatives comprising gonochoristic, hermaphroditic, and parthenogenetic reproductive strategies (Poinar, 1983). Morphologically, the spermatozoa in the entire group are characterized by the absence of a flagellum and an acrosome as well as the presence of membranous vesicles (Justine, 2002). To acquire motility, a spherical spermatid must first undergo the process known as spermiogenesis or sperm activation, in which a pseudopod is extended, conferring motility to the cell in an amoeboid fashion (see Fig. 1B). This process of acquisition of motility has been most extensively studied in the nematodes Ascaris suum and Caenorhabditis elegans, and is currently thought to be the general mode of sperm locomotion in all nematodes. Although, most studies on these sperm have been performed using molecular genetics and biochemistry, an integrative physiological framework is lacking. The present study is intended to summarize the physiological pathways that lead to the in vitro extension of the pseudopod in nematode sperm from both Ascaris and Caenorhabditis. IN VITRO PSEUDOPOD EXTENSION AND CRAWLING OF NEMATODE SPERMATOZOA Nematode spermatozoa initiate motility subsequent to the extension of the pseudopod (in the case of C. elegans, or lamellipod in A. suum) allowing the cell to crawl on a substrate. A peculiarity in these amoeboid cells is that they contain non-actin-based microfilaments responsible for pseudopod extension (Nelson et al., 1982). Instead of actin, nematode sperm form filaments from the Major Sperm Protein (MSP), a 14 kDa protein that constitutes 15% of the total protein in the sperm (Klass and Hirsh, 1981). To polymerize, MSP must be present in dimers to elongate filaments and fibers allowing the cell to crawl (Roberts and Stewart, 2000). Despite the threefold difference in maximum length between the spermatozoa of C. elegans (∼9 µm) and A. suum (∼26 µm; Royal et al., 1997), the process of pseudopod extension in both nematodes is very similar and initiates with rearrangements of the plasma membrane (Nelson and Ward, 1980) and the extension of transient membrane protrusions known as “spikes” in C. elegans or filipodia in A. suum. Filaments of MSP form these “spikes,” which precede the formation of the pseudopod in both A. suum and C. elegans (Shakes and Ward, 1989; Rodriguez et al., 2005). Thus, for the purpose of this review, we refer to the physiological signals that initiate membrane rearrangements and spike protrusions as sperm activation, a process that results in pseudopod extension and the consequent acquisition of motility. Although the molecule(s) responsible for in vivo sperm activation in nematodes are not known to date, the process of in vitro sperm activation has been studied extensively in both species. In the case of A. suum, the sperm extends a crescent-shaped flat pseudopod with distinguishable MSP fiber bundles (Sepsenwol et al., 1989). Spermatids can be activated in vitro using either S. griseus proteases (25 µg/ml) or a vas deferens extract (VDX) from A. suum (Sepsenwol and Taft, 1990). Sperm activated using proteases extend a pseudopod, but are unable to crawl, while activation with VDX induces crawling at an average instant velocity of 30.3 ± 16.2 µm/min (Royal et al., 1997). In contrast, spermatozoa from C. elegans possess a less-flat pseudopod that lacks visible MSP fiber bundles. Spermatids are activated in vitro using either S. griseus proteases (200 µg/ml), Triethanolamine (TEA), a weak base that promotes an increase in intracellular pH (Ward et al., 1983), or the cationic ionophore Monensin (Nelson and Ward, 1980). C. elegans spermatozoa can crawl in vitro at an average instantaneous velocity of 12.0 ± 4.9 µm/min (Royal et al., 1997), similar to the reported in vivo average velocity of 8.07 ± 0.67 µm/min of male spermatozoa crawling in the hermaphrodite uterus (Kubagawa et al., 2006). Protease-activated spermatozoa from C. elegans are also unable to crawl. PLASMA MEMBRANE DYNAMICS AND MICRODOMAINS The first step in the process of sperm activation is the rearrangement of the plasma membrane. Roberts and Ward (1982b) observed this dramatic rearrangement by attaching latex beads to the plasma membrane of C. elegans spermatids and, using a microscope, followed their movements throughout the activation process. This experiment revealed that the plasma membrane of spermatids undergoes an intermittent, nondirected movement at a rate of 10–15 µm/min on discrete portions of the cell surface. This movement, coupled to cell rotation, initiates after treatment with Monensin, stops once the pseudopod is completely formed, and is not present in mutant sperm that fail to extend a pseudopod (Roberts and Ward, 1982b). The extension of “spike” structures in nematode sperm coincides with membrane rearrangements and a highly fluid cell surface, resulting in the dynamic steps of protrusion, retraction, thickening, and fusion of spikes to coalesce into a pseudopod or lamellipod (Shakes and Ward, 1989; Rodriguez et al., 2005). After the extension of the pseudopod and before the acquisition of motility, the nematode-specific Membranous Organelles (MOs) localize to the periphery of the cell body and fuse to the plasma membrane, delivering proteins and membrane material necessary for the total extension of the pseudopod, motility acquisition and successful fertilization (L'Hernault, 2006; Singson et al., 2008). At the time of MOs fusion, all the membrane rearrangements cease in the cell body while the initiation of directed membrane flow from the tip to the base of the pseudopod confers motility to the cell (Roberts and Ward, 1982a). Protein insertion has been observed at the leading edge of the pseudopod, although it is not clear how this process occurs as there are no organelles found in this region and no endocytosis processes or vesicle trafficking has been reported (Roberts and Ward, 1982a; Pavalko and Roberts, 1987; Shakes and Ward, 1989). Membrane Microdomains In mammalian spermatozoa, capacitation (the collective physiological processes that render sperm competent for fertilization) accompanies the remodeling of membrane microdomains that are responsible for the acrosome reaction, sperm–egg recognition, and the ultimate fusion of the sperm and egg plasma membranes (Gadella et al., 2008; Nixon and Aitken, 2009). In other cell types, these cholesterol-enriched dynamic microdomains, often referred to as lipid rafts, are proposed to functionally cluster membrane proteins within common intracellular pathways (Simons and Toomre, 2000; Golub et al., 2004). Recently, this view of “patchy” and regionalized membranes has been discussed (Engelman, 2005). In amoeboid sperm, the plasma membrane is a very dynamic structure that must maintain the integrity of interactions between proteins in common signaling pathways. Thus, we speculate that the integrity of protein interactions is maintained due to the presence of membrane microdomains as compelling evidence supports this hypothesis for C. elegans sperm (see Fig. 2). For instance, the SPE-8 group of proteins (that include SPE-8, SPE-12, SPE-19, SPE-27, and SPE-29), responsible as a trigger for spike formation and further pseudopod extension, is thought to function as a multicomponent complex that involves the interaction of membrane and cytoplasmic signaling proteins (Geldziler et al., 2005; see Fig. 2A). Other membrane microdomains could be hypothesized in the docking and fusion of MOs to the plasma membrane (see Fig. 2B) as FER-1, a protein involved in vesicle fusion, has a human homolog that interacts with Caveolin-3 (Matsuda et al., 2001), a membrane microdomain marker. FER-1 is hypothesized to promote vesicle fusion through protein–protein interactions with SNARE proteins (Washington and Ward, 2006), a complex localized in cholesterol-enriched microdomains (Lang, 2007). Figure 2Open in figure viewerPowerPoint 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 filament 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. A supporting argument on the presence of membrane microdomains is revealed by cholesterol localization in spermatids and the protein redistribution that occurs during pseudopod extension. Matyash et al. (2001) used a cholesterol fluorescent analog, dehydroergosterol, to localize its distribution in C. elegans during development, showing that males displayed a strong labeling in dispersed cytoplasmic structures of the spermatid. Also in spermatids, proteins that are important for fertilization change their localization subsequent to the extension of the pseudopod (Singson et al., 2008). For instance, SPE-9 is an EGF repeat transmembrane protein localized homogeneously over the spermatid plasma membrane, while after pseudopod extension it is found exclusively on the pseudopod (Zannoni et al., 2003). 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 SPE-41 (Xu and Sternberg Paul, 2003), and the MO marker 1CB4 (Okamoto and Thomson, 1985). All of these are localized to MOs prior to pseudopod extension and change their distribution upon fusion to the plasma membrane. SPE-38 co-localizes to the pseudopod with SPE-9, TRP-3 redistributes to the plasma membrane in both the cell body and the pseudopod, and the 1CB4 marker is maintained in the MOs bodies (see Fig. 2B). In Ascaris sperm, there is also evidence for membrane microdomains. MSP nucleation and elongation occurs at the leading edge of the pseudopod where the MSP Polymerization Organizing Protein (MPOP) recruits cytosolic proteins to the membrane surface (LeClaire et al., 2003). Elongation and bundling of the filaments into fibers occurs at the leading edge of the pseudopod, forming a complex intertwined network of MSP (Roberts and Stewart, 2000; Bottino et al., 2002). This cytoskeletal network is connected mechanically to the substratum through the membrane, allowing directional movement of the cell. In C. elegans sperm, cells crawl by traction exerted through plasma membrane proteins on the substrate. This attachment can be inhibited by antibodies directed against the membrane proteins TR11 and SP56, but not by the anti MSP antibody, TR20 (Pavalko et al., 1988). Altogether, these results lead to the hypothesis that MSP is localized to the cytoplasmic leaflet of the plasma membrane and other proteins involved in attachment and traction are localized to the extracellular leaflet. If these proteins are co-localized in discrete regions of attachment we can speculate that crawling in nematode sperm might be similar to the formation of focal adhesions in cells with an actin-based motility where ordered membrane microdomains play an important role (Wozniak et al., 2004; Gaus et al., 2006; see Fig. 2C). Collectively, this body of information leads us to hypothesize that membrane microdomains are present in nematode sperm and involved in the processes of sperm activation, MO fusion to the plasma membrane, and motility acquisition, rendering the cell competent for successful fertilization. Thus, future experiments in nematode sperm motility can be designed to test whether proteins that are redistributed in the plasma membrane may be released or sequestered within different microdomains and localize to specific morphological regions (in an analogous fashion with mammalian spermatozoa) such as the pseudopod, where they can move from the tip to the base, thereby recycling receptors for sperm–egg fusion and conferring attachment of the cell to the substrate resulting in successful fertilization. ION PHYSIOLOGY AND SIGNAL TRANSDUCTION DURING PSEUDOPOD EXTENSION In flagellated spermatozoa, membrane rearrangements are hypothesized to have an effect on the biophysical properties of the plasma membrane with an impact on ion channel and/or enzymatic activity on the cell surface (Kopf et al., 1999). These changes in ionic fluxes are required for an increase in sperm motility and to achieve capacitation through plasma membrane hyperpolarization, alkalization of intracellular pH, increased intracellular Ca2+ concentrations, and the initiation of intracellular signaling cascades that involve protein phosphorylation events (for reviews see Darszon et al., 2008; Suarez, 2008; Abou-haila and Tulsiani, 2009). In nematode sperm, ionic fluxes during pseudopod extension have been studied to a lesser extent (Fig. 3). Machaca et al. (1996) used the patch clamp technique to investigate voltage-sensitive ion channel activities in C. elegans sperm and calculated the spermatid's resting potential (VR = −8.07 ± 2.26 mV) in Nystatin-perforated whole-cell experiments. These investigators discovered several voltage-sensitive ion channel activities in both spermatocytes and residual bodies during spermatogenesis, although only an inward-rectifying chloride channel (Clir) was detected in spermatids upon hyperpolarization. In the same study, the chloride channel inhibitor 4,4′-di-isocyanatostilbene-2,2′-disulfonic acid was found to induce activation in spermatids, suggesting an important role of Clir channels in the process of pseudopod extension. Sperm activation by the blockage of Clir is suggested to act by either a change in membrane potential or by a transport of HCO ions into the cytoplasm, resulting in an increase of intracellular pH (Machaca et al., 1996). Indeed, the use of ionophores such as Monensin and Valinomycin has provided insight on changes in sperm intracellular pH necessary to maintain pseudopod extension in both C. elegans and A. suum (Nelson and Ward, 1980; Roberts and King, 1991). However, this cytosolic alkalization is coupled to the exchange of protons with Na+, in the case of Monensin, and K+, in the case of Valinomycin, making it unclear whether spermatid activation is coupled to cytosolic alkalization in general or to specific changes in Na+ or K+ ions. In this regard, it has been shown that both ions are necessary for in vitro pseudopod extension as triethanolamine and Monensin spermatid activation are reduced or abolished when Na+ and K+ are replaced in the medium (Nelson and Ward, 1980; Ward et al., 1983). Activation with Pronase is also reduced when K+ ions are replaced, although this activator does not alter intracellular pH (Ward et al., 1983). Nelson and Ward (1980) also suggested a trigger for pseudopod extension due to changes in membrane potential based on K+-dependent mechanisms (Nelson and Ward, 1980). To our knowledge, the mechanisms for how nematode sperm membrane potential is affected during pseudopod extension using any in vitro activator have not been investigated to date. Figure 3Open in figure viewerPowerPoint Ionic conductance plays an important role in the acquisition of motility in amoeboid sperm. Although studied to a lesser extent, ion fluxes are necessary for pseudopod extension and motility maintenance. The blockage of Clir channels induces sperm activation due to changes in the resting membrane potential and/or the accumulation (or transport) of HCO into the cytoplasm, causing alkalization. Na+ and K+ ions are necessary for sperm activation and could also affect membrane potential. An alternative is that the exchange of Na+ and 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. Intracellular pH As is the case in flagellated sperm, an increase in intracellular pH is necessary for motility acquisition in nematode sperm. This was first suggested when spermatids treated with Monensin showed a pH dependence on pseudopod extension (Nelson and Ward, 1980). Weak bases, including TEA, also exerted pseudopod extension accompanied by an increase in intracellular pH (Ward et al., 1983). In this study, the authors reported that cytoplasmic alkalization is sufficient to trigger spermatid activation and that removal of TEA did not affect pseudopod morphology. However, in A. suum spermatozoa, treatment with weak acids caused loss of motility, disassembly of the MSP cytoskeleton and, consequently, compromised pseudopod integrity (Roberts and King, 1991). Thus, maintenance of a specific intracellular pH, putatively buffered by HCO, is crucial for pseudopod integrity and crawling of spermatozoa. Further, the MSP cytoskeleton polymerization involved in amoeboid movement (protrusion and retraction) is regulated by an intracellular pH gradient along the pseudopod (King et al., 1994; Italiano et al., 1999). Using the pH-sensitive fluorescent indicator BCECF, King et al. (1994), demonstrated an intracellular alkalization of 0.25 pH units during activation of spermatids using vas deferens extract (VDX). The weak bases, TEA and NH4Cl, induced a similar pH increase of 0.21 and 0.32 units respectively, although, only blebs or “spikes” were formed by the spermatid and no pseudopod was extended. In this study, they also showed an increase of 0.15 pH units between the tip of the pseudopod where alkalization correlates with MSP fibers assembly, and the base of the pseudopod where acidification promotes disassembly of the MSP cytoskeleton. Thus, mechanisms of intracellular pH regulation are involved in the trigger and maintenance of motility forces in amoeboid sperm (Bottino et al., 2002). Intracellular Ca2+ Ca2+ is a key second messenger that regulates sperm capacitation and motility in flagellated sperm together with Ca2+-binding proteins (Abou-haila and Tulsiani, 2009). In nematode sperm, the role of Ca2+ during motility acquisition has only been studied in C. elegans; however, these studies yield insights not only on the physiological stages of Ca2+ signaling during pseudopod extension but also on the conserved role of this ion during exocytosis and fertilization. In 1980, Nelson and Ward demonstrated that pseudopod extension is not induced with either of the Ca2+ ionophores A23187 or X537A. External Ca2+ is not necessary either for the trigger of spermatid activation with any of the in vitro activators, Monensin, TEA or Pronase, since removal of this ion from the medium has little or no effect on pseudopod extension (Nelson and Ward, 1980; Ward et al., 1983). Intriguing results from Shakes and Ward in 1989 showed that the Calmodulin inhibitors Trifluoperazine (TFP), Chlorpromazine (CPZ), and Naphtalenesulfonamide (W7) can induce pseudopod extension, although the pseudopod is abnormal in that it lacks villar projections and is devoid of membrane movement (Shakes and Ward, 1989). Once the Calmodulin inhibitor was removed, the pseudopod morphology and movement recovered normally. The Ca2+ channel blocker nicardipine was reported in the same article to have a similar effect. These investigators also observed that spermatids from the spe-8 and spe-12 mutants arrest in the “spike” stage after activation with Pronase. This spike arrest could be overcome and pseudopod extension occurred following treatment with TFP, and the other Calmodulin inhibitors, although TEA and Monensin had the same effect. All of these observations were reported to occur in the absence of external Ca2+, suggesting the involvement of internal Ca2+ stores. Furthermore, it was shown that motile spermatozoa treated with the Calmodulin inhibitors stop motility and rounding up of the pseudopod. Shakes and Ward (1989) were not able to detect Calmodulin in spermatids using antibodies that cross-react with nematode Calmodulin. Together, this evidence complemented with the known effects of TFP, CPZ, and W7 on processes unrelated to Calmodulin, led the authors to conclude as unlikely the effect of these drugs on the inhibition of Calmodulin, as this would suggest a rather odd role of blocking pseudopod extension by this Ca2+-binding protein, given that Calmodulin in flagellated sperm plays an important role in hyperactivation of motility, capacitation, and acrosome reaction through protein tyrosine phosphorylation events. Subsequently, the Ca2+-binding protein, Calreticulin (CRT-1), was observed to be present in the cytoplasm of C. elegans sperm. The crt-1 mutant spermatozoa appeared to have slightly shorter pseudopods and nuclei that were off center, suggesting a role for CRT-1 in the late stages of spermatogenesis (Park et al., 2001). The same research group discovered the presence of Calcineurin, a Ca2+/Calmodulin-dependent serine/threonine protein phosphatase (PP2B) in C. elelgans sperm, with cnb-1 mutants sharing the same phenotype as crt-1 mutant sperm – short pseudopod and reduced size (Bandyopadhyay et al., 2002). Whether, Calmodulin, or a Calmodulin-dependant mechanism, regulates Ca2+ dynamics during pseudopod extension is still unclear, although Ca2+-regulated machinery is certainly present in C. elegans spermatids. This Ca2+-dependent machinery is complemented by FER-1, a protein of the ferlin family involved in the Ca2+-mediated MO fusion to the plasma membrane (Washington and Ward, 2006). FER-1 is characterized by multiple C2 domains that function as Ca2+ sensors and interact with phospholipids and proteins of the membrane fusion machinery during exocytosis (Bai and Chapman, 2004). It has also been demonstrated that intracellular Ca2+ stores are involved in the proper fusion of MOs to the plasma membrane since the membrane-permeable Ca2+ chelator BAPTA-AM prevented MO fusion in a concentration-dependent manner (Washington and Ward, 2006). In Drosophila melanogaster, the ferlin gene misfire (mfr) is a homolog of C. elegans fer-1, and is involved in early embryogenesis and egg patterning in females, and in the completion of fertilization in males (Ohsako et al., 2003; Smith and Wakimoto, 2007). Successful fertilization in D. melanogaster involves sperm entry (by puncturing a hole in the egg oolemma without the involvement of membrane fusion) followed by sperm plasma membrane break down (PMBD), nuclear decondensation, and the formation of a male pronucleus. A testis isoform of Mfr containing five C2 domains and a transmembrane domain is required for sperm PMBD, a process also mediated by Ca2+-dependent membrane interactions between the acrosome and the plasma membrane (Smith and Wakimoto, 2007). The final piece of evidence for Ca2+ dynamics during pseudopod extension involves a Transient Receptor Potential channel (TRPC) homolog, TRP-3, present in C. elegans spermatozoa and required for sperm–egg interaction during fertilization (Xu and Sternberg Paul, 2003). As explained previously, TRP-3 is localized