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Biology of Reproduction 67, 1043-1051 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Control of Membrane Fusion During Spermiogenesis and the Acrosome Reaction1

João Ramalho-Santosc, Gerald Schattenb, and Ricardo D. Moreno2,a

a Unit of Reproduction and Development, Physiology Department, Pontifical Catholic University of Chile, 340-213 Santiago, Chile b Pittsburgh Development Center of Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213 c Center for Neuroscience and Cell Biology, Department of Zoology, University of Coimbra, 3004-517 Coimbra, Portugal


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 INTRACELLULAR MEMBRANE FUSION...
 MOLECULAR EVENTS DURING ACROSOME...
 MEMBRANE FUSION DURING THE...
 PERSPECTIVES
 REFERENCES
 
Membrane fusion is important to reproduction because it occurs in several steps during the process of fertilization. Many events of intracellular trafficking occur during both spermiogenesis and oogenesis. The acrosome reaction, a key feature during mammalian fertilization, is a secretory event involving the specific fusion of the outer acrosomal membrane and the sperm plasma membrane overlaying the principal piece of the acrosome. Once the sperm has crossed the zona pellucida, the gametes fuse, but in the case of the sperm this process takes place through a specific membrane domain in the head, the equatorial segment. The cortical reaction, a process that prevents polyspermy, involves the exocytosis of the cortical granules to the extracellular milieu. In lower vertebrates, the formation of the zygotic nucleus involves the fusion (syngamia) of the male pronucleus with the female pronucleus. Other undiscovered membrane trafficking processes may also be relevant for the formation of the zygotic centrosome or other zygotic structures. In this review, we focus on the recent discovery of molecular machinery components involved in intracellular trafficking during mammalian spermiogenesis, notably related to acrosome biogenesis. We also extend our discussion to the molecular mechanism of membrane fusion during the acrosome reaction. The data available so far suggest that proteins participating in the intracellular trafficking events leading to the formation of the acrosome during mammalian spermiogenesis are also involved in controlling the acrosome reaction during fertilization.

gamete biology, gametogenesis, sperm, spermatid, spermatogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
INTRACELLULAR MEMBRANE FUSION...
 MOLECULAR EVENTS DURING ACROSOME...
 MEMBRANE FUSION DURING THE...
 PERSPECTIVES
 REFERENCES
 
The acrosome reaction (AR) is the exocytic event by which the acrosome fuses with the sperm plasma membrane and exposes its contents to the extracellular milieu [1]. Like regulated secretion in many other systems, the AR is triggered by an increase in intracellular calcium. However, the molecular events that take place after the calcium surge and that lead to actual fusion between the outer acrosomal membrane and the sperm plasma membrane remain obscure [2].

Much is known regarding the molecular mechanisms of regulated exocytosis in secretory cells, and many of the factors that modulate this process also participate in membrane trafficking between cytoplasmic organelles [3]. However, morphological studies have revealed that the process of acrosome biogenesis shares many characteristics with the formation of exocytic vesicles in secretory cells [4–6]. Thus, by studying the molecular fusion machinery components involved in mammalian acrosome biogenesis we may gain new insights into the mechanism of AR. In this review, we summarize exciting new data concerning the molecular fusion machinery present during mammalian acrosome formation. The proteins present in the acrosome of mature sperm may also play a role in membrane fusion in the biogenesis of the acrosome during mammalian spermiogenesis.


   INTRACELLULAR MEMBRANE FUSION MACHINERY
TOP
 ABSTRACT
 INTRODUCTION
 INTRACELLULAR MEMBRANE FUSION...
MOLECULAR EVENTS DURING ACROSOME...
 MEMBRANE FUSION DURING THE...
 PERSPECTIVES
 REFERENCES
 
Membrane flow in somatic cells follows an elaborate choreography that must be tightly regulated to ensure that transport vesicles fuse with their appropriate targets and that cargo molecules are delivered to the correct sites. Many proteins regulate different transport events at various levels, and although the picture is by no means complete, the details of a complex system are beginning to emerge.

Initial studies in membrane trafficking identified an N-ethylmaleimide sensitive factor (the ATPase NSF) as having an important role in intracellular fusion [7, 8]. NSF exerts its activity via the recruitment of soluble NSF attachment proteins (SNAPs, such as alpha- and beta-SNAP) [8]. However, these components are ubiquitous in many fusion events and cannot be used to determine specificity. This paradox was partially resolved with the discovery of a large family of membrane-bound SNAP receptors (SNAREs) [8]. The localization of different SNAREs in different cellular compartments is thought to ensure that a transport vesicle docks/fuses with its appropriate target and not with any other membrane [9, 10]. At a functional level, this SNARE model postulates that fusion between two membranes takes place following the interaction of one or more SNAREs on the membrane of the transport vesicle (v-SNAREs) with a complement of one or more SNAREs on the target membrane (t-SNAREs), although this localization may not be exclusive (i.e., some v-SNAREs may exist on the target membrane, and some t-SNAREs may exists on the vesicle) [11]. Pairing of t- and v-SNAREs would result in an energetically favored complex, bringing the two membranes into close contact and leading to fusion. The v- and t-SNARE were defined initially in heterotypic fusion events (i.e., fusion of two different membranes), and this classification is therefore less relevant in homotypic fusion (i.e., fusion of identical membranes) [12, 13]. For this reason, SNAREs are also often classified structurally, as Q-SNAREs and R-SNAREs, depending on the presence of a conserved glutamine or arginine on the helix that is involved in SNARE complex formation [14]. A SNARE complex between two fusion partners (termed a trans complex) is usually formed by a bundle of four helices containing three Q coils and one R coil [15]. In synaptic vesicle fusion to the plasma membrane, the best characterized system, the Q helices are provided by the t-SNAREs syntaxin 1 (one) and SNAP-25 (two) on the plasma membrane, whereas the R helix in on the v-SNARE VAMP-1 on the synaptic vesicle [16, 17]. However, this equivalency between Q- and t-SNAREs, on one hand, and R- and v-SNAREs, on the other, is not a general rule.

Using synaptic vesicle exocytosis as an experimental model for the study of regulated secretion, it was also found that SNAREs can interact with calcium channels on the plasma membrane, and this interaction may be important for triggering effective neurotransmitter exocytosis after a signaling calcium surge [18, 19]. Other molecules associated with the SNARE complex, such as synaptotagmins (sometimes also classified as v-SNAREs), may serve as calcium sensors in these fusion events. Synaptotagmins could act by "clamping" the SNARE complex until the intracellular calcium concentration increases and membrane merging can take place [18–21]. Therefore, after the calcium surge, synaptotagmin or synaptotagmin-like molecules probably activate SNARE-induced membrane fusion events.

Pairing of SNAREs is sufficient to drive (slow) membrane merging in reconstituted systems, namely using recombinant SNAREs that have been inserted into pure lipid bilayers (liposomes) [22]. Recent work suggests that at least in some cases the final steps may be mediated by other factors, such as calmodulin, protein phosphatase 1, and pore-forming complexes such as the V0 subunit of H+-ATPases [14, 23, 24]. However, the current viewpoint postulates that NSF and SNAPs act to disentangle SNARE interactions that may have formed within a single membrane (cis complexes) and thus free them to interact with SNAREs in another membrane [25]. This ATP-dependent chaperone activity, termed priming, may also be important in disrupting complexes and recycling SNAREs after fusion has taken place. The list of possible SNARE regulators continues to grow at a steady pace [26]. The issue is further confounded by recent developments, which suggest that rather than relying on absolute specificity the SNARE interactions may actually be promiscuous, with each SNARE protein potentially involved in several pairings [27, 28].

Upstream from the trans SNARE complex, other molecules are thought to be involved in transporting vesicles across the cytoplasm, bringing them in contact with their target membranes and aiding in the formation of the correct SNARE pairings. The tethering factors are characterized by their large size (>250 kDa) and in general adopt a rodlike structure in solution [29]. Among these factors are proteins such as giantin, GM130, and p115, which participate in the transport of cargo proteins between the endoplasmic reticulum and the Golgi apparatus [30–33]. The precise role of these molecules is still under debate, but it seems that they participate in the very first steps of vesicle-membrane recognition [29]. Crucial among docking factors is the growing family of small GTPases termed rabs. Like other GTPases, rabs cycle between a GDP-bound (on) and a GTP-bound (off) conformation, but superimposed on this cycle there is another regulatory process [34]. Rabs can exist either as a cytoplasmic or a membrane-bound form. Although these two cycles are not exactly equivalent, rab association with membranes (via acylation) usually occurs in the GTP-bound form [29, 34]. Pinpointing the specific role of rab proteins in membrane fusion has been quite an elusive task. However, it is clear that specific isoforms, such as rab5 and rab7, are essential for the homotypic fusion of early and late endosomes, respectively [35–38]. In addition, rabs may coordinate the assembly of the tethering/docking complex during vesicle budding. Recent work suggests that rab1 controls the association of the tethering protein p155 with the SNAREs syntaxin 5 and rbet1 in the early secretory pathway [39]. In this context, rabs could bestow identity and control the specificity of vesicle targeting. Rabs are in turn regulated by a myriad of other factors.


   MOLECULAR EVENTS DURING ACROSOME BIOGENESIS
TOP
 ABSTRACT
 INTRODUCTION
 INTRACELLULAR MEMBRANE FUSION...
 MOLECULAR EVENTS DURING ACROSOME...
MEMBRANE FUSION DURING THE...
 PERSPECTIVES
 REFERENCES
 
Proacrosomal Granule Formation and Assembly of the Acrosome

In humans, rhesus monkeys, and mice, the synthesis of many acrosome-specific proteins, such as proacrosin and acrogranin, begins at the pachytene spermatocyte stage [40, 41]. These proteins follow the exocytic pathway, and are packed in electron-dense core vesicles called proacrosomal granules (PAGs), probably at the trans-Golgi level. These granules do not proceed to the plasma membrane but instead remain in the boundaries of the Golgi apparatus until the completion of meiosis and the ensuing formation of the round spermatid [5, 41, 42]. PAGs have a diameter of about 500 nm, and their number may vary among species [43]. Because these granules are formed by pachytene spermatocytes and they will not fuse to each other until the completion of meiosis, there must be some mechanism to prevent precocious fusion of these vesicles. Whether this process involves the synthesis or elimination of specific components from the PAGs is an open issue. Alternatively, an inductor of the fusion and/or translocation of the PAGs may be activated in round spermatids. Although many aspects of pachytene spermatocyte meiosis are well known, namely reagarding chromosome interactions and recombination, the mechanisms responsible for PAG packaging and assembly in these cells remain unexplored. Additionally, the biosynthetic route taken by species such as cattle, pigs, and rats is different, because in these species the proacrosin gene is expressed in the round spermatid and not in spermatocytes [44]. Nonetheless, the PAGs are formed at equivalent steps of differentiation as described for humans and mice.

The formation and assembly of the acrosomal vesicle begins at step 1 spermatids (Fig. 1). Initially, PAGs are close to the concave face of the Golgi apparatus, but then they start to translocate to the nuclear surface, clustering together [5, 43, 45]. Step 3 spermatids contain only one acrosomal vesicle, which is assumed to result from fusion of the individual PAGs, although there is no direct evidence to support this hypothesis (Fig. 1) [5, 43, 45]. At this step, the nascent acrosome is attached to the nuclear envelope or to the spermatid perinuclear theca [46]. Because PAGs are formed by pachytene spermatocytes, there must be a mechanism that prevents precocious fusion of these vesicles. We have shown in rhesus monkeys and mice that syntaxin and the v-SNARE VAMP envelop the PAGs of stage-1 and stage-2 spermatids [5, 47]. At later steps of differentiation, VAMP and syntaxin localize to the acrosomal vesicle and remain there until the mature sperm is formed. Are these SNAREs active in the fusion process or merely being recruited for later action during the AR? Or are there different members of the SNARE family responsible for these distinct events? The other major issue is that we do not know the specific isoforms localized at this step of development. A differential expression of t- or v-SNAREs in PAGs of pachytene spermatocytes and early spermatids may control the initial steps of acrosome formation. In other systems, specific isoforms of syntaxin and VAMP are localized at specific domains along the exocytic secretory pathway, and they may regulate the fusogenic capability of different membranous compartments [26]. We do not know which isoform is been expressed during mammalian spermiogenesis and whether it is conserved, even among different species. In this context, syntaxin 2 has been localized to the acrosomal region in rat spermatozoa [48]. Whether this isoform is expressed in other mammals or is unique to the rat, remains to be established.



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  		FIG. 1. During spermiogenesis three major developmental processes occur: 1) the condensation of the nucleus, 2) the formation of the flagellum, and 3) the formation of the acrosome. Early round spermatids (1) have a round nucleus (blue), marked by the presence of a large Golgi complex and several proacrosomal vesicles. The flagellum has started to differentiate and lies near the Golgi area. Later on (2), the PAGs fuse to each other and form the acrosomal vesicle over the nucleus, which starts to grow because of fusion of Golgi-derived vesicles. The flagellum moves toward the opposite site of the acrosome, near the nucleus. One of the most dramatic transformations during spermiogenesis happens when the acrosomal-nucleus complex turns upside down and moves toward the plasma membrane (3). During this process the Golgi apparatus migrates toward the opposite pole of the nascent acrosome, and the acrosomal vesicle lies underneath the plasma membrane (4, 5). The Golgi apparatus remains in the cytoplasmic lobe and, along with other cytoplasmic organelles, is discarded in the cytoplasmic droplet. In the final steps of differentiation, the acrosomal vesicle flattens and spreads over the nucleus, covering up to two-thirds of its surface. Some selected mitochondria (white ovals) move to the sperm flagellum, and others are discarded in the cytoplasmic droplet. Concomitant with all these events, the nucleus reaches its final shape, and the sperm is released to the lumen of the seminiferous tubule

The process of acrosome biogenesis shares many characteristics with the formation of secretory granules in neuroendocrine cells, just as the AR shares similarities with secretory exocytosis in somatic cells [49]. Secretory granules originate from the trans-Golgi network (TGN) with a selected subset of luminal and membrane proteins, and at this stage they are named immature secretory granules (ISGs) [49]. Heterotrimeric G proteins regulate the biogenesis of ISGs, although how these factors participate in sorting during ISGs formation is not known [49]. The differences between ISGs and mature secretory granules (MSGs) in both size and content have led to the proposal that the nascent ISGs are an intermediate stage in secretory granule biogenesis [13]. Like PAGs, maturation of the intermediate ISGs to MSGs requires both fusion and budding events [13]. The maturation period involves the selective retrieval of proteins that are not present in the MSGs, such as furin, lysosomal enzymes, and the mannose-6-phosphate receptor (M6PR) [50]. The sorting of these proteins occurs via vesicles positive for adaptor protein 1, clathrin, and syntaxin 6 [51, 52]. In addition, during this process there is a progressive acidification of the intravesicular pH from ISGs (pH 6.3) to MSGs (pH 5.5) [49]. These coordinated actions should exert a tight control on both the composition and size of secretory granule content that is released upon stimulation.

Mammalian acrosome biogenesis shares some similarities with secretory granule formation. As for ISGs, the acrosomal vesicle (and probably PAG) is able to incorporate the acidothropic probe LysoTracker DND-26 [4, 5]. We do not know if there are any changes in the intra-acrosomal pH during spermiogenesis, but the acrosomal vesicle is sufficiently acid to capture the dye throughout the process. LysoTracker has been used to label the acrosome of bull and rhesus monkey sperms and to label lysosomes in somatic cells [5, 53, 54]. However, there is no report concerning the labeling of the Golgi apparatus with this dye in somatic cells. We have hypothesized that the probe probably labels the trans-Golgi apparatus or the TGN, because these are the most acid subcompartments of the organelle in somatic cells. Because LysoTracker has been used to label lysosomes in somatic cells, and given that lysosomes have a significantly lower pH than the Golgi apparatus, it is possible that the Golgi label has been obscured by the more intense contribution of lysosomes. Alternatively, the Golgi apparatus of spermatids may have a lower pH than the equivalent organelle in somatic cells, thus allowing a higher uptake of the dye. The lower pH of the Golgi apparatus in spermatids may reflect a significant difference between spermatogenic and somatic cells. This characteristic may be related to the unique timing and mechanisms involved in sorting of acrosomal proteins during spermiogenesis.

Growth and Shaping of the Acrosomal Vesicle: Role of Membrane Trafficking and the Golgi Apparatus

During steps 4–7 in the mouse or equivalent steps in other species, the acrosomal vesicle grows and then flattens over the spermatid nucleus, covering up to two-thirds of the total nuclear surface [45, 55]. Electron microscopy data have suggested that the acrosomal vesicle's growth in volume is mainly due to the constant fusion of Golgi-derived vesicles [43, 56]. Nevertheless, there is no evidence that the vesicles found between the Golgi stacks and the acrosome are going in any particular direction (Fig. 2). In addition, many convolutions and blebs on the acrosomal membrane of guinea pigs have been interpreted as proof of vesicle fusion [57]. These figures may in fact represent early steps of pinched-off vesicles rather than late fusion steps. It has been proposed that the PAGs form as a result of the progressive fragmentation of the trans-Golgi saccules, which peel off the trans-face of the stacks [58]. Scanning electron microscopy of rat spermatids indicates that the anastomosing TGN breaks down into strings of connected vesicles, which arise from the edge of the saccules in stage-5 spermatids [58]. Therefore, the acrosomal vesicle would grow due to the fusion of whole Golgi stacks or cisternae, rather than vesicles. If the spermatids use this later mechanism to accomplish acrosomal growth, some Golgi resident proteins should be present in the acrosome.



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  		FIG. 2. Molecular model of mammalian acrosome formation during spermiogenesis. A) In mammals, the acrosome-specific proteins follow the exocytic pathway and are packed in electron-dense core vesicles (PAGs) probably at the trans-Golgi level. The PAGs are formed by distinct areas: an electron-dense core containing acrosomal area (dark dot) and a pale area that fades away during later steps of spermiogenesis. The acrosome-specific proteins are located in the dense core, which will become the acrosomal matrix in the mature sperm. We envision two alternative mechanisms of PAG and acrosome formation, where the PAGs come off the Golgi apparatus individually (1) or attached to each other like beads in a string (2). In either way, the PAGs will move from the Golgi apparatus to the nuclear envelope (NE) carrying acrosomal enzymes (dense core) and Golgi proteins, in the pale region, such as giantin or mannosidase II. Once the PAGs have attached to the NE, the acrosome vesicle starts to grow. The current hypothesis states that Golgi-derived vesicles contribute to this process. We propose that these vesicles, along with whole cistenae of the Golgi (2), fuse with the growing acrosome. In this way, the growth of the acrosome vesicle may be 2-fold, from Golgi-derived membranes and from membranes derived from the lysosome-endosome system. B) The lysosome-endosome system. There is no information about the precise role of early endosomes (EE), late endosomes (LE), or lysosomes (L) during spermiogenesis and how these compartments manage the membrane flow between them. According to our model, the mammalian acrosome may be considered one member of this system, because it shares some targeting proteins (e.g., rab5 and rab7) with EE and LE. In this context, some membrane traffic could occur from endosomes to the acrosomal vesicle (AV) or vice versa (white arrows)

Immunofluorescence and immunogold studies carried out with probes against cis and medial Golgi proteins, such as Golgin95/GM130, giantin, mannosidase II, and beta-COP, revealed a horseshoe-shaped Golgi apparatus, with the concave side facing the acrosome, in steps 2–7 of rhesus monkey, mouse, and bull spermatids [4, 5, 55]. Beta-COP, a protein involved in vesicular transport from the Golgi apparatus to the endoplasmic reticulum and between Golgi stacks, is present at the rims of the Golgi cisternae in developing bull spermatids [55]. Moreover, all of these probes are also found in the acrosomal vesicle in step 2 to step 7 spermatids (Figs. 1 and 2) but not in late elongating spermatids or mature sperm in mouse, bull, or rhesus monkey [4, 5, 47, 55]. However, this fact may represent a completely new mechanism of secretory vesicle formation, where Golgi stacks fall apart and fuse with the immature vesicle. This membrane flow may help to remodel the composition of the acrosomal membrane and may somehow participate in the shaping of the acrosome. A number of lysosomal proteins detected in the mouse acrosomal vesicle are also subsequently retrieved throughout differentiation [59, 60]. Mannosidase IA resides in the rat acrosome of spermatids up to step 15 [61, 62]. The mechanism for removal of these proteins may involve a specific degradation pathway at late steps of spermiogenesis. Some enzymes of the ubiquitin-mediated degradation pathway are present within the acrosome of developing spermatids [63]. However, vesicles coated with clathrin and probably those coated with beta-COP might participate in the retrieval of missorted proteins from the acrosome. These proteins are present in late elongating spermatids, suggesting a membrane remodeling process of the acrosome at that stage [55]. Such a remodeling of vesicular membranes also takes place in ISGs, where syntaxin 6 and M6PR along with other nonspecific proteins are removed during maturation [52]. Despite this retrieval mechanism, the acrosome of mature sperm contains both unique acrosomal enzymes and common enzymes of lysosomal origin [62]. Their presence in the acrosome may reflect a low efficiency of the spermatid sorting machinery, and the SNARE and rab families of proteins could regulate this process. Syntaxin, VAMP, and NSF are present in the acrosomal vesicle throughout acrosome biogenesis and in the mature organelle found in human and bull sperm [5, 6, 47, 55]. However, although alpha- and beta-SNAPs have been detected in the developing acrosome [47], they have yet to be characterized in mature sperm. Whether the same SNARE isoforms are present during the entire process or there is a selective stage-specific expression of SNAREs during spermiogenesis remains to be resolved.

Important regulators of membrane trafficking are the members of the rab family of small GTPases [64, 65]. These proteins play a crucial role in making sure that transport vesicles fuse with their appropriate targets [34, 65]. Rab6 has been found mainly on the Golgi apparatus and as a faint acrosomal label in mouse developing spermatids [47]. Its presence on the acrosome may therefore reflect retrograde retrieval of Golgi proteins from the acrosome using this pathway. However, rab5-positive vesicles are found in close proximity to the acrosome, notably in step 4 and step 5 spermatids, and rabaptin-5, a downstream effector for rab5, has a similar pattern of distribution [47]. Rab7 was also found associated mainly with the acrosomal vesicle in both mouse and bull developing spermatids [47, 66]. Rab3A, thought to modulate the AR, was detected only in the acrosome of epididymal sperm and not in earlier steps of differentiation [47, 66]. These new and exciting findings open the possibility of an unexpected contribution of the endosome-lysosome system to the formation of the acrosome, which is particularly interesting in the light of recent work on secretory lysosomes, the calcium-dependent fusogenic activity of which is controlled by synaptotagmins (Fig. 2) [20]. Synaptotagmins have been found in both human and mouse sperm, although the isoform present in each species seems to be different (Table 1) [6, 67, 68]. Nonetheless, lysosomal (lgp-120) or late endosomal (M6PR) markers appear to be associated solely with lysosomes in rat spermatids and not with the acrosome or proacrosomal vesicles [69]. Therefore, the acrosome seems to be a distinct and complex new vesicle, with an identity that is different from any organelle/vesicle in other exocytic or endocytic systems. However, it remains to be determined whether the presence of either early or late endosomal markers has a functional consequence in the fusogenic capability of the acrosomal vesicle with the different compartments of the endocytic system [66]. In somatic cells, rab5 and rab7 regulate the docking and homotypic fusion of early and late endocytic vesicles, respectively [37, 53]. Rab7 also regulates the heterotypic fusion of late endosomes with lysosomes and the homotypic fusion of lysosomes [37, 53]. Thus, traffic through the endocytic pathway to the lysosome seems to be controlled by the extent of homotypic and heterotypic fusion among the different vesicular compartments.


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  		TABLE 1. Proteins participating in intracellular trafficking and their localization in different species

In mouse spermatids at step 7 (or equivalent in other species), the acrosome-nucleus complex moves towards the cell surface and remains attached to the plasma membrane [70]. As a result of this process, the acrosome-nucleus complex rotates so that the acrosome faces the basal membrane rather than the lumen of the seminiferous tubule (Fig. 1). This process generates two clear domains in the acrosome membrane that will be of prime importance during the AR. The part of the acrosome membrane lining the nuclear surface is the inner acrosomal membrane (IAM), and that attached to the plasma membrane is the outer acrosomal membrane (OAM) [70]. The OAM fuses with the plasma membrane during the AR in capacitated sperm, but the IAM remains intact even after the sperm fuses with the oocyte plasma membrane [2, 71]. Thus, these two membrane domains are functionally, and probably molecularly, different. There is no information about the genesis of these differences and how they are maintained over time, but they probably arise during spermiogenesis. One enticing hypothesis is that by step 7 the domains of the acrosomal membrane are already set up and the OAM has SNARE proteins that guide the acrosomal-nucleus complex in its route thru the plasma membrane. In this way, proteins such as VAMP, syntaxin, or rab could specify the target membrane of the acrosomal-nucleus complex (Fig. 2). The same proteins may have some role during the next step in the biology of the sperm, the AR. By this period of differentiation, there is an extensive rearrangement of the microtubule cytoskeleton [72, 73]. In addition, new structures appear, such as the manchette, that have specific isoforms of tubulin and may have specific functions in this process [72–74]. In this way, the reordering of the cytoskeleton could help in the traffic and reorientation of the acrosomal-nucleus complex up to the plasma membrane, similarly to what has been described in somatic cells [75].


   MEMBRANE FUSION DURING THE AR
TOP
 ABSTRACT
 INTRODUCTION
 INTRACELLULAR MEMBRANE FUSION...
 MOLECULAR EVENTS DURING ACROSOME...
 MEMBRANE FUSION DURING THE...
PERSPECTIVES
 REFERENCES
 
The mammalian AR is a very special case of regulated secretion. As in many other such events, exocytosis of vesicular contents into the extracellular milieu takes place after a signaling mechanism (triggered by sperm binding to the egg's zona pellucida or by other stimuli) invokes an increase in intracellular calcium [76]. However, not only is the vesicle enlarged at this time (especially with respect to the spermatozoon itself), but fusion takes place at several distinct points of contact and results in the destruction and concomitant shedding of the fusing partners [71, 77]. Another characteristic of the mammalian AR is the seemingly slow, controlled exposure of the vesicular contents, possibly to better aid the spermatozoon in its gradual penetration of the egg's investments [78–80]. This release is likely to be regulated by the dense acrosomal matrix. The action of fusion may not be an immediate all-or-nothing phenomenon but may occur in different acrosomal regions at different time points [71, 78]. The AR may also be modulated by changes in the spermatic membrane and by cytoskeletal elements such as the F-actin bundles that exist below the plasma membrane, which may act as steric inhibitors of membrane contact [81]. The success of the AR relies upon the capacitation state of the sperm. Capacitation is a process of sperm maturation unique to mammals, which may happen either in vivo or in vitro and has been related to many intracellular and extracellular changes [82, 83]. Only capacitated sperm can respond to natural inducers of the AR, such as progesterone and the egg's zona pellucida proteins [82, 83]. The molecular changes involved in the priming of the outer acrosomal membrane and plasma membrane prior to fusion during the AR are not understood.

Is membrane fusion in this system regulated by SNAREs? Are there SNAREs on the acrosome? A preliminary answer to the latter question has been recently obtained, with the discovery that both syntaxin and VAMP exist on the acrosomal region of sea urchin and many types of mammalian sperm, although their exact cellular location (i.e., on the plasma membrane or on the outer acrosomal membrane) has not been unequivocally ascertained (Fig. 3) [84, 85]. Our group was the first to obtain evidence that both syntaxin and VAMP are localized on the acrosome of mammalian sperm and that antibodies against these proteins inhibited the AR [6]. Thereafter, syntaxin 2 was localized in the acrosome of rat sperm and recently syntaxins 1A, 1B, 4, and 6 were found in the acrosome of human sperm [48, 86]. In addition, VAMP2, SNAP-25, and SNAP-23 are also present in human sperm [86]. The SNAREs SNAP-23 and SNAP-25 are not related to the NSF cofactor SNAPs (such as alpha- and beta-SNAP) that have been found in the developing acrosome [47]. The mechanism used by SNAREs to induce the AR appears similar to a process that occurs in neuronal cells [9, 10, 18, 25, 26, 65, 86]. In this context, a massive increase in the intracellular calcium concentration would trigger the assembly of the syntaxin, VAMP, and SNAP complex (Fig. 3). Synaptotagmin, a protein that supposedly senses the intracellular calcium concentration, has been detected on sperm heads of hamsters, mice, bulls, rhesus monkeys, and humans [6]. The probes used in this initial study were directed against SNAREs that participate in synaptic vesicle exocytosis, namely syntaxin 1, synaptotagmin I, and VAMP-1 and -2, but cross-reactivity with other close family members is always a possibility [6, 47]. In subsequent work, syntaxin 2 was detected on the head of mouse sperm [48]. In this context, it seems that synaptotagmin VI is located on the head of human sperm and synaptotagmin VIII is located in mouse sperm [67, 68]. Additional evidence indicates that synaptotagmin VII is also present in the acrosome of mouse spermatozoa (unpublished results). Whether this finding represents a species-specific difference or cross-reactivity of the antibodies or probes used in each case needs to be clarified. Regardless, SNAREs are now known to be present on the mammalian acrosomal region, with evidence suggesting that they may indeed mediate membrane merging during the AR (Table 1 and Fig. 2).



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  		FIG. 3. A putative role of SNAREs during the mammalian AR. The AR is the exocytic event by which the acrosome fuses with the sperm plasma membrane and exposes its contents to the extracellular milieu. Crucial among docking factors is the growing family of small GTPases termed rabs, which cycle as cytoplasmic (rab-GDP) or membrane-bound (rab-GTP) forms. A) In our model an extracellular signal, such as ZP3, would induce the activation and binding of sperm rab proteins (rab3A) with its effectors. B) This complex would in turn activate the ATPase NSF, inducing the release of the SNARE blocker complex from the SNAREs. C) In our model, we propose that t-SNAREs (yellow) and v-SNAREs (black) are in the plasma membrane and OAM, respectively. The t-SNAREs located in the plasma membrane would be in a high molecular weight complex associated with a calcium channel and a sperm receptor. D) Like regulated secretion in many other systems, the AR is triggered by an increase in intracellular calcium, which is probably released from internal stores via the production of inositol 1,4,5-triphosphate. Other molecules associated with the SNARE complex, such as synaptotagmin (red) or calmodulin, may serve as calcium sensors in these fusion events. In this way, these molecules will free the v-SNARE (black) at the outer acrosomal membrane, allowing the the interaction with the t-SNARE (yellow) at the plasma membrane. E) Releasing of the SNARE blocker complex, and the calcium sensor proteins, will enable the pairing of t- and v-SNAREs, bringing the two membranes into close contact and leading to the fusion of both membranes

SNAREs are by no means the only molecules on the acrosome that could regulate membrane merging. NSF (but not SNAPs) has been described on the mature acrosome in human sperm [87] (Table 1). However, the putative calcium sensor (synaptotagmin) had been described in humans [6, 68], mice [6, 67], rhesus monkeys [6], bulls [6], and hamsters [6], although the specific isoform found in each species seems to be different (Table 1). Although the picture is not yet complete, the need for rabs and NSF during the AR suggests that these proteins may be playing roles similar to those they play in somatic cells. Therefore, rab3A may participate in docking/tethering of the OAM to the sperm plasma membrane, an event that must therefore take place late in sperm maturation, possibly even during capacitation [27, 34]. However, NSF could break up possible cis SNARE complexes, allowing for fusion-triggering trans connections, because destruction of the fusion partners following the AR implies that recycling of SNAREs probably does not take place. Of course, these molecules may act in a totally novel way during the AR, and more work is needed to establish the proper sequence of events. An intriguing observation is that the rab3A knockout mouse, although displaying some neurologic impairment, is fertile [88, 89]. In addition, the rabphilin (one of the rab5 effectors) knockout mouse is also fertile and does not seem to have any major morphologic or functional alterations [90]. Thus, neither rab3A nor rabphillin appear to be essential to sperm biogenesis, and the spermatid might use alternative pathways in their absence. However, we do not know whether gamete interactions, egg activation, or embryo development are altered in those knockout mice. A similar suggestion was raised when sperm lacking acrosin, the sperm protease, fertilized zona-intact eggs in vitro [91]. However, the fertilization rate of these sperm was slower than that of control wild type sperm, suggesting that some sperm components may not be essential but do improve sperm function. This issue is important because fertilization is a process where the best and fastest sperm wins the right to fertilize the oocyte [82].


   PERSPECTIVES
TOP
 ABSTRACT
 INTRODUCTION
 INTRACELLULAR MEMBRANE FUSION...
 MOLECULAR EVENTS DURING ACROSOME...
 MEMBRANE FUSION DURING THE...
 PERSPECTIVES
REFERENCES
 
As is usual with new approaches to established problems, the discovery of membrane trafficking machinery components on the developing and mature acrosome has raised more intriguing and fertile questions than it has answered. Although many components (rabs, SNAREs, NSF) may participate in membrane fusion during both acrosome biogenesis and the AR, these events are by no means equivalent. Thus, during spermiogenesis the acrosome is targeted to the nucleus and fusion (of PAGs stored by the spermatocyte) is initially homotypic, with possible late contributions from other compartments. However, the AR implies the targeting of the organelle to the plasma membrane and concomitant heterotypic fusion, which could involve, for example, a switch from rab5- and rab7-mediated tethering during spermiogenesis to rab3A action in mature cells. Other issues concern the nature of the trafficking molecules themselves. What is the final tally of SNAREs and rabs on the acrosome? Are there any testis-specific forms? Where exactly are they located, and does their location change during spermiogenesis, capacitation, or the AR? Does the point-by-point fusion process that characterizes the AR imply a discontinuous SNARE localization at either the plasma membrane or acrosomal membrane, or are other factors involved? Although SNAREs are uniformly distributed on the synaptic plasma membrane, synaptic vesicle fusion only occurs at certain locations, suggesting the need for other regulatory elements. More importantly, which molecules described so far have functionally relevant activity and at what levels? What is the sequence of action? Is there any species specificity? Are there built-in redundancy mechanisms that ensure that the once-in-a-lifetime fusion event that is the AR takes place even if one element is missing or nonfunctional, such as in the rab3A and rabphilin knockout mice? These and many other tantalizing questions ensure a busy future for researchers.


   ACKNOWLEDGMENTS
 
This work is dedicated to our dear friend Prof. Dr. Claudio Barros, who dedicated his life to understanding the acrosome reaction and, in doing so, made friends all over the world.


   FOOTNOTES
 
1 This work was supported by research grants from DIPUC (2001/05E) and CONRAD (MFG-00-56) to R.D.M and NIH (NICHD, NCRR) to G.S. J.R.-S. was the recipient of a PRAXIS XXI postdoctoral fellowship from Fundação para a Ciência e Tecnologia (FCT, Portugal), and received additional support from Fundação Luso-Americana para o Desenvolvimento (FLAD, Portugal). He is currently supported by a research grant from FCT (POCTI/ESP/38049/2001). Back

2 Correspondence: Ricardo D. Moreno, Unit of Reproduction and Developmental Biology, Physiology Department, Faculty of Biological Sciences, Pontifical Catholic University of Chile, 340-213 Santiago, Chile. FAX: 56 2 222 5515; rmoreno{at}genes.bio.puc.cl Back

Received: 3 April 2002.

First decision: 23 April 2002.

Accepted: 25 April 2002.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 INTRACELLULAR MEMBRANE FUSION...
 MOLECULAR EVENTS DURING ACROSOME...
 MEMBRANE FUSION DURING THE...
 PERSPECTIVES
 REFERENCES
 

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