The dynamics of SNARE assembly and disassembly during membrane recognition and fusion is a central issue in intracellular trafficking and regulated secretion. Exocytosis of sperm's single vesicle—the acrosome—is a synchronized, all-or-nothing process that happens only once in the life of the cell and depends on activation of both the GTP-binding protein Rab3 and of neurotoxin-sensitive SNAREs. These characteristics make acrosomal exocytosis a unique mammalian model for the study of the different phases of the membrane fusion cascade. By using a functional assay and immunofluorescence techniques in combination with neurotoxins and a photosensitive Ca2+ chelator we show that, in unactivated sperm, SNAREs are locked in heterotrimeric cis complexes. Upon Ca2+ entry into the cytoplasm, Rab3 is activated and triggers NSF/α-SNAP-dependent disassembly of cis SNARE complexes. Monomeric SNAREs in the plasma membrane and the outer acrosomal membrane are then free to reassemble in loose trans complexes that are resistant to NSF/α-SNAP and differentially sensitive to cleavage by two vesicle-associated membrane protein (VAMP)–specific neurotoxins. Ca2+ must be released from inside the acrosome to trigger the final steps of membrane fusion that require fully assembled trans SNARE complexes and synaptotagmin. Our results indicate that the unidirectional and sequential disassembly and assembly of SNARE complexes drive acrosomal exocytosis.

Exocytosis of the acrosome (the acrosome reaction) relies on cAMP production, assembly of a proteinaceous fusion machinery, calcium influx from the extracellular medium, and mobilization from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Addition of cAMP to human sperm suspensions bypasses some of these requirements and elicits exocytosis in a protein kinase A-and extracellular calcium-independent manner. The relevant cAMP target is Epac, a guanine nucleotide exchange factor for the small GTPase Rap. We show here that a soluble adenylyl cyclase synthesizes the cAMP required for the acrosome reaction. Epac stimulates the exchange of GDP for GTP on Rap1, upstream of a phospholipase C. The Epac-selective cAMP analogue 8-pCPT-2-O-Me-cAMP induces a phospholipase Cdependent calcium mobilization in human sperm suspensions. In addition, our studies identify a novel connection between cAMP and Rab3A, a secretory granule-associated protein, revealing that the latter functions downstream of soluble adenylyl cyclase/cAMP/Epac but not of Rap1. Challenging sperm with calcium or 8-pCPT-2-O-Me-cAMP boosts the exchange of GDP for GTP on Rab3A. Recombinant Epac does not release GDP from Rab3A in vitro, suggesting that the Rab3A-GEF activation by cAMP/Epac in vivo is indirect. We propose that Epac sits at a critical point during the exocytotic cascade after which the pathway splits into two limbs, one that assembles the fusion machinery into place and another that elicits intracellular calcium release.

The acrosome reaction is a unique type of regulated exocytosis. The single secretory granule of the sperm fuses at multiple points with the overlying plasma membrane. In the past few years we have characterized several aspects of this process using streptolysin O-permeabilized human spermatozoa. Here we show that Rab3A triggers acrosomal exocytosis in the virtual absence of calcium in the cytosolic compartment. Interestingly, exocytosis is blocked when calcium is depleted from intracellular stores. By using a membrane-permeant fluorescent calcium probe, we observed that the acrosome actually behaves as a calcium store. Depleting calcium from this compartment by using a light-sensitive chelator prevents secretion promoted by Rab3A. UV inactivation of the chelator restores exocytosis. Rab3A-triggered exocytosis is blocked by calcium pump and inositol 1,4,5-trisphosphate (IP 3 )-sensitive calcium channel inhibitors. Calcium measurements inside and outside the acrosome showed that Rab3A promotes a calcium efflux from the granule. Interestingly, release of calcium through IP 3 -sensitive calcium channels was necessary even when exocytosis was initiated by increasing free calcium in the extraacrosomal compartment in both permeabilized and intact spermatozoa. Our results show that a calcium efflux from the acrosome through IP 3 -sensitive channels is necessary downstream Rab3A activation during the membrane fusion process leading to acrosomal exocytosis.The acrosome reaction is an exocytotic process induced physiologically by the activation of sperm receptors by ligands in the zona pellucida of the oocyte. This interaction initiates a complex transduction mechanism leading to multiple fusions between the outer acrosomal membrane and the overlying plasmalemma resulting in the release of the acrosomal content and exposure of the molecules present on the inner acrosomal membrane.As in other regulated secretory events, calcium plays a central role in acrosome reaction (1). Recent results from different laboratories suggest that a first transient cytosolic calcium increase (probably mediated by T-type calcium channels (2)) leads to a second sustained increase of cytosolic calcium that is necessary for the acrosome reaction (3-9). Although the connection between the two events is not completely clear, the current hypothesis is that the first calcium increase causes the activation of a phospholipase C (PLC).1 There are several isoforms of PLC in the spermatozoa (10, 11). PLC␦4, in particular, has been implicated in the early events of the acrosome reaction (12). Active PLC would produce inositol 1,4,5-trisphosphate (IP 3 ) that would open IP 3 -sensitive calcium channels in the membrane of intracellular stores. The emptying of these stores would trigger the opening of store-operated calcium (SOC) channels in the plasma membrane causing a second and sustained calcium increase that would trigger acrosomal exocytosis (1, 7). Although direct proof for the nature of the calcium stores involved in this mechanism is lacking, severa...

Epac, a guanine nucleotide exchange factor for the small GTPase Rap, binds to and is activated by the second messenger cAMP. In sperm, there are a number of signaling pathways required to achieve egg-fertilizing ability that depend upon an intracellular rise of cAMP. Most of these processes were thought to be mediated by cAMP-dependent protein kinases. Here we report a new dependence for the cAMP-induced acrosome reaction involving Epac. The acrosome reaction is a specialized type of regulated exocytosis leading to a massive fusion between the outer acrosomal and the plasma membranes of sperm cells. Ca 2؉ is the archetypical trigger of regulated exocytosis, and we show here that its effects on acrosomal release are fully mediated by cAMP. Ca 2؉ failed to trigger acrosomal exocytosis when intracellular cAMP was depleted by an exogenously added phosphodiesterase or when Epac was sequestered by specific blocking antibodies. The nondiscriminating dibutyrylcAMP and the Epac-selective 8-(p-chlorophenylthio)-2-O-methyladenosine-3,5-cyclic monophosphate analogues triggered the acrosome reaction in the effective absence of extracellular Ca 2؉ . This indicates that cAMP, via Epac activation, has the ability to drive the whole cascade of events necessary to bring exocytosis to completion, including tethering and docking of the acrosome to the plasma membrane, priming of the fusion machinery, mobilization of intravesicular Ca 2؉ , and ultimately, bilayer mixing and fusion. cAMP-elicited exocytosis was sensitive to anti-␣-SNAP, anti-NSF, and anti-Rab3A antibodies, to intra-acrosomal Ca 2؉ chelators, and to botulinum toxins but was resistant to cAMP-dependent protein kinase blockers. These experiments thus identify Epac in human sperm and evince its indispensable role downstream of Ca 2؉ in exocytosis.

SummaryThe acrosome is a single secretory granule present in the head of mammalian -and other animal groups -sperm. Secretion of this granule is an absolute requirement for physiological fertilization. Acrosome exocytosis is a synchronized and tightly regulated all-or-nothing process, with no recycling of membranes. In the last few years, it has been shown that acrosomal exocytosis is mediated by a molecular mechanism that is homologous to that reported in the secretion of neuroendocrinal cells. Moreover, because of its particular characteristics, acrosomal exocytosis is a unique mammalian model for the study of the different steps of the membrane fusion cascade. Combining results in intact and permeabilized sperm, the following sequence of events has been proposed. In resting sperm, SNARE proteins are locked in inactive cis complexes. Sperm activation causes a calcium increase in the cytoplasm that promotes the production of cAMP and activates Rab3A. Afterwards, NSF and aSNAP disassemble cis complexes and the free SNAREs are then able to reassemble in loose trans complexes. Membrane fusion is arrested at this stage until calcium is released from inside the acrosome by inositol 1,4,5-trisphosphate-sensitive calcium channels to trigger the final steps of membrane fusion, which require fully assembled trans SNARE complexes and the calcium sensor synaptotagmin. This working model is still incomplete and tentative. Its improvement will be important to share light on this and other processes of regulated exocytosis. Moreover, it will bring new perspectives into the field of sperm-related fertility and sterility.

The acrosome reaction is a regulated exocytotic process leading to a massive fusion between the outer acrosomal membrane and the cell membrane. In spite of the great amount of information available related to the acrosome reaction in several species, there is a remarkable paucity about the role of monomeric guanosine triphosphatases (GTPases) of the Rab family-well-established participants in exocytosis in other cell types-in the acrosome reaction. Western blot and immunofluorescence analysis indicate that Rab3A is present in human spermatozoa and localizes to the acrosomal region in the sperm head. One difficulty in studying the role of proteins in intact cells is the fact that they are unable to cross the cell membrane. Therefore, we established a working model of streptolysin O-permeabilized human spermatozoa. Permeabilized spermatozoa were able to respond in a regulated way to different stimuli, such as G protein activators and calcium. An acrosomal reaction was also triggered by a Rab3A peptide corresponding to the effector region. More important, recombinant Rab3A protein in the GTP-bound form caused acrosome exocytosis. The same protein loaded with GDP or Rab11 in the GTP-bound form was inactive. Also, recombinant GDI (GDP dissociation inhibitor)-a protein that releases Rab proteins from membrane-inhibited a GTPgammaS-stimulated acrosome reaction. Our results indicate that 1) permeabilized spermatozoa can be used to study the role of macromolecules in the acrosome reaction, 2) Rab3A is present in human spermatozoa, and 3) Rab3A or another Rab3 isoform is involved in the exocytosis of the acrosomal granule in human spermatozoa.

Regulated secretion is a fundamental process underlying the function of many cell types. In particular, acrosomal exocytosis in mammalian sperm is essential for egg fertilization. Regulated secretion requires SNARE proteins and, in neurons, also synaptotagmin I and complexin. Recent reports suggest that complexin imposes a fusion block that is released by Ca 2؉ and synaptotagmin I. However, no direct evidence for this model in secreting cells has been provided and whether this complexin/ synaptotagmin interplay functions in other types of secretion is unknown. In this report, we show that the C2B domain of synaptotagmin VI and an anti-complexin antibody blocked the formation of trans SNARE complexes in permeabilized human sperm, and that this effect was reversed by adding complexin. In contrast, an excess of complexin stopped exocytosis at a later step, when SNAREs were assembled in loose trans complexes. Interestingly, this blockage was released by the addition of the synaptotagmin VI C2B domain in the presence of Ca 2؉ . We have previously demonstrated that the activity of this domain is regulated by protein kinase C-mediated phosphorylation. Here, we show that a phosphomimetic mutation in the polybasic region of the C2B domain strongly affects its Ca 2؉ and phospholipids binding properties. Importantly, this mutation completely abrogates its ability to rescue the complexin block. Our results show that the functional interplay between complexin and synaptotagmin has a central role in a physiological secretion event, and that this interplay can be modulated by phosphorylation of the C2B domain.All types of intracellular membrane fusions, including regulated exocytosis (i.e. the stimulus-triggered fusion of secretory vesicles with the plasma membrane), require proteins from the SNARE 3 family (1). SNAREs are small membrane proteins that form stable hetero-oligomeric complexes consisting of a bundle of four parallel helices. Assembly of trans SNARE complexes between SNAREs from two opposing membranes is a key event for membrane fusion. SNAREs residing on the same membrane can form cis complexes that must be disassembled to render free SNAREs competent for membrane fusion. The exocytotic SNAREs involved in neurotransmission are syntaxin1A and SNAP25 in the plasmalemma, and synaptobrevin 2 (also called VAMP2) in secretory vesicles. These proteins are the targets of botulinum and tetanus toxins, a set of highly specific zinc-dependent endoproteases (2). Only when not assembled in tight complexes are SNAREs susceptible to cleavage, making these toxins excellent tools for the diagnosis of SNARE assembly status.Additional proteins that are key for Ca 2ϩ -triggered neurotransmitter release include complexins and synaptotagmins (3). Complexins are small cytosolic proteins that bind with high affinity to SNARE complexes (4), forming an antiparallel ␣ helix that inserts into a groove between the synaptobrevin and syntaxin helices (5). Experiments where complexins were deleted or overexpressed have suggested that they play ...

Exocytosis of the acrosome (the acrosome reaction) is a terminal morphological alteration that sperm must undergo prior to penetration of the extracellular coat of the egg. Ca(2+) is an essential mediator of this regulated secretory event. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, NSF, and synaptotagmin VI in the human sperm acrosome reaction. Interestingly, Rab3A elicits an exocytotic response of comparable magnitude to that of Ca(2+). Here, we report a direct role for the SNARE complex in the acrosome reaction. First, the presence of SNARE proteins is demonstrated by Western blot. Second, the Ca(2+)-triggered acrosome reaction is inhibited by botulinum neurotoxins BoNT/A, -E, -C, and -F. Third, antibody inhibition studies show a requirement for SNAP-25, SNAP-23, syntaxins 1A, 1B, 4, and 6, and VAMP 2. Fourth, addition of bacterially expressed SNAP-25 and SNAP-23 abolishes exocytosis. Acrosome reaction elicited by Rab3-GTP is also inhibited by BoNT/A, -C, and -F. Taken together, these results demonstrate a requirement for members of all SNARE protein families in the Ca(2+)- and Rab3A-triggered acrosome reaction. Furthermore, they indicate that the onset of sperm exocytosis relies on the functional assembly of SNARE complexes.