Enhanced Expansion and Reduced Kiss-and-Run Events in Fusion Pores Steered by Synaptotagmin-1 C2B Domains.

The fusion pore controls the release of exocytotic vesicle contents through a precise orchestration of lipids from the fusing membranes and proteins. There is a major lipid reorganization during the different stages in life of the fusion pore (membrane fusion, nucleation, and expansion) that can be scrutinized thermodynamically. In this work, using umbrella sampling simulations we describe the expansion of the fusion pore. We have calculated free energy profiles to drive a nascent, just nucleated, fusion pore to its expanded configuration. We have quantified the effects on the free energy of one and two Synaptotagmin-1 C2B domains in the cytosolic space. We show that C2B domains cumulatively reduce the cost for expansion, favoring the system to evolve toward full fusion. Finally, by conducting thousands of unbiased molecular dynamics simulations, we show that C2B domains significantly decrease the probability of kiss-and-run events.

The Synaptotagmin-1 C2B Domain Is a Key Regulator in the Stabilization of the Fusion Pore.

Fusion pores serve as an effective mechanism to connect intracellular organelles and release vesicle contents during exocytosis. A complex lipid rearrangement takes place as membranes approximate, bend, fuse, and establish a traversing water channel to define the fusion pore, linking initially isolated chambers. Thermodynamically, the process is unfavorable and thought to be mediated by specialized proteins. In this work, we have developed a reaction coordinate to induce fusion pores from initially flat and parallel lipid bilayers and we have used it to describe the effects of the synaptotagmin-1 C2B domain during the process. We have obtained free-energy profiles of the whole lipid reorganization in biologically realistic membranes, going from planar and parallel bilayers through stalk hemifusion to water channel formation. Our results point to a lysine-rich polybasic region on synaptotagmin-1 C2B as the key to lipid reorganization control through the formation of phosphatidylinositol bisphosphate clusters that stabilize the fusion pore.

Reactive oxygen species are involved in the signaling of equine sperm chemotaxis.

Sperm chemotaxis may facilitate the finding of the oocyte. Only capacitated spermatozoa can orient their movement by chemotaxis, which as well as capacitation, is regulated in part by the cAMP-PKA pathway. Reactive oxygen species (ROS) are produced during sperm capacitation which is closely related to chemotaxis. Then, the ROS participation in the chemotactic signaling can be expected. Here we studied the role of ROS in the chemotaxis signaling of equine spermatozoa which produce high quantities of ROS because of their energy metabolism. The level of capacitated and chemotactic spermatozoa was increased with 0.1 and 0.2 mM hydrogen peroxide (H2O2), which was involved in the chemotactic signaling. By combining a concentration gradient of H2O2 with inhibitors/chelators of some of the signaling pathway elements, we showed that the activation of NOX (membrane NADPH oxidase) increases the intracellular ROS which activate the chemotaxis AMPc-PKA pathway. Our results provide evidence about the participation of ROS in the chemotactic signaling mediated by progesterone (P).

Membrane-permeable Rab27A is a regulator of the acrosome reaction: Role of geranylgeranylation and guanine nucleotides.

The acrosome reaction is the regulated exocytosis of mammalian sperm's single secretory granule, essential for fertilization. It relies on small GTPases, the cAMP binding protein Epac, and the SNARE complex, among other components. Here, we describe a novel tool to investigate Rab27-related signaling pathways: a hybrid recombinant protein consisting of human Rab27A fused to TAT, a cell penetrating peptide. With this tool, we aimed to unravel the connection between Rab3, Rab27 and Rap1 in sperm exocytosis and to deepen our understanding about how isoprenylation and guanine nucleotides influence the behaviour of Rab27 in exocytosis. Our results show that TAT-Rab27A-GTP-γ-S permeated into live sperm and triggered acrosomal exocytosis per se when geraylgeranylated but inhibited it when not lipid-modified. Likewise, an impermeant version of Rab27A elicited exocytosis in streptolysin O-permeabilized - but not in non-permeabilized - cells when geranylgeranylated and active. When GDP-ß-S substituted for GTP-γ-S, isoprenylated TAT-Rab27A inhibited the acrosome reaction triggered by progesterone and an Epac-selective cAMP analogue, whereas the non-isoprenylated protein did not. Geranylgeranylated TAT-Rab27A-GTP-γ-S promoted the exchange of GDP for GTP on Rab3 and Rap1 detected by far-immunofluorescence with Rab3-GTP and Rap1-GTP binding cassettes. In contrast, TAT-Rab27A lacking isoprenylation or loaded with GDP-ß-S prevented the activation of Rab3 and Rap1 elicited by progesterone. Challenging streptolysin O-permeabilized human sperm with calcium increased the population of sperm with Rap1-GTP, Rab3-GTP and Rab27-GTP in the acrosomal region; pretreatment with anti-Rab27 antibodies prevented the activation of all three. The novel findings reported here include: the description of membrane permeant TAT-Rab27A as a trustworthy tool to unveil the regulation of the human sperm acrosome reaction by Rab27 under physiological conditions; that the activation of endogenous Rab27 is required for that of Rab3 and Rap1; and the connection between Epac and Rab27 and between Rab27 and the configuration of the SNARE complex. Moreover, we present direct evidence that Rab27A's lipid modification, and activation/inactivation status correlate with its stimulatory or inhibitory roles in exocytosis.

The Rab3A-22A Chimera Prevents Sperm Exocytosis by Stabilizing Open Fusion Pores.

At the final stage of exocytotis, a fusion pore opens between the plasma and a secretory vesicle membranes; typically, when the pore dilates the vesicle releases its cargo. Sperm contain a large dense-core secretory granule (the acrosome) whose contents are secreted by regulated exocytosis at fertilization. Minutes after the arrival of the triggering signal, the acrosomal and plasma membranes dock at multiple sites and fusion pores open at the contact points. It is believed that immediately afterward, fusion pores dilate spontaneously. Rab3A is an essential component of human sperm exocytotic machinery. Yet, recombinant, persistently active Rab3A halts calcium-triggered secretion when introduced after docking into streptolysin O-permeabilized cells; so does a Rab3A-22A chimera. Here, we applied functional assays, electron and confocal microscopy to show that the secretion blockage is due to the stabilization of open fusion pores. Other novel findings are that sperm SNAREs engage in α-SNAP/NSF-sensitive complexes at a post-fusion stage. Complexes are disentangled by these chaperons to achieve vesiculation and acrosomal contents release. Thus, post-fusion regulation of the pores determines their expansion and the success of the acrosome reaction.

The Molecules of Sperm Exocytosis.

Exocytosis is a fundamental process used by eukaryotic cells to release biological compounds and to insert lipids and proteins in the plasma membrane. Specialized secretory cells undergo regulated exocytosis in response to physiological signals. Sperm exocytosis or acrosome reaction (AR) is essentially a regulated secretion with special characteristics. We will focus here on some of these unique features, covering the topology, kinetics, and molecular mechanisms that prepare, drive, and regulate membrane fusion during the AR. Last, we will compare acrosomal release with exocytosis in other model systems.

The signaling module cAMP/Epac/Rap1/PLCε/IP3 mobilizes acrosomal calcium during sperm exocytosis.

Exocytosis of the sperm's single secretory granule, or acrosome, is a regulated exocytosis triggered by components of the egg's investments. In addition to external calcium, sperm exocytosis (termed the acrosome reaction) requires cAMP synthesized endogenously and calcium mobilized from the acrosome through IP3-sensitive channels. The relevant cAMP target is Epac. In the first part of this paper, we present a novel tool (the TAT-cAMP sponge) to investigate cAMP-related signaling pathways in response to progesterone as acrosome reaction trigger. The TAT-cAMP sponge consists of the cAMP-binding sites of protein kinase A regulatory subunit RIß fused to the protein transduction domain TAT of the human immunodeficiency virus-1. The sponge permeated into sperm, sequestered endogenous cAMP, and blocked exocytosis. Progesterone increased the population of sperm with Rap1-GTP, Rab3-GTP, and Rab27-GTP in the acrosomal region; pretreatment with the TAT-cAMP sponge prevented the activation of all three GTPases. In the second part of this manuscript, we show that phospholipase Cε (PLCε) is required for the acrosome reaction downstream of Rap1 and upstream of intra-acrosomal calcium mobilization. Last, we present direct evidence that cAMP, Epac, Rap1, and PLCε are necessary for calcium mobilization from sperm's secretory granule. In summary, we describe here a pathway that connects cAMP to calcium mobilization from the acrosome during sperm exocytosis. Never before had direct evidence for each step of the cascade been put together in the same study.

Small GTPases in acrosomal exocytosis.

Regulated exocytosis employs a conserved molecular machinery in all secretory cells. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Rab superfamilies are members of this machinery. Rab proteins are small GTPases that organize membrane microdomains on organelles by recruiting specific effectors that strongly influence the movement, fusion and fission dynamics of intracellular compartments. Rab3 and Rab27 are the prevalent exocytotic isoforms. Many events occur in mammalian spermatozoa before they can fertilize the egg, one of them is the acrosome reaction (AR), a type of regulated exocytosis. The AR relies on the same fusion machinery as all other cell types, which includes members of the exocytotic SNARE and Rab superfamilies. Here, we describe in depth two protocols designed to determine the activation status of small G proteins. One of them also serves to determine the subcellular localization of active Rabs, something not achievable with other methods. By means of these techniques, we have reported that Rab27 and Rab3 act sequentially and are organized in a RabGEF cascade during the AR. Although we developed them to scrutinize the exocytosis of the acrosome in human sperm, the protocols can potentially be extended to study other Ras-related proteins in virtually any cellular model.

Epac, Rap and Rab3 act in concert to mobilize calcium from sperm's acrosome during exocytosis.

BACKGROUND: Exocytosis of sperm's single secretory granule or acrosome (acrosome reaction, AR) is a highly regulated event essential for fertilization. The AR begins with an influx of calcium from the extracellular milieu and continues with the synthesis of cAMP and the activation of its target Epac. The cascade bifurcates into a Rab3-GTP-driven limb that assembles the fusion machinery and a Rap-GTP-driven limb that mobilizes internal calcium. RESULTS: To understand the crosstalk between the two signaling cascades, we applied known AR inhibitors in three experimental approaches: reversible, stage-specific blockers in a functional assay, a far-immunofluorescence protocol to detect active Rab3 and Rap, and single cell-confocal microscopy to visualize fluctuations in internal calcium stores. Our model system was human sperm with their plasma membrane permeabilized with streptolysin O and stimulated with external calcium. The inhibition caused by reagents that prevented the activation of Rap was reversed by mobilizing intracellular calcium pharmacologically, whereas that caused by AR inhibitors that impeded Rab3's binding to GTP was not. Both limbs of the exocytotic cascade joined at or near the stage catalyzed by Rab3 in a unidirectional, hierarchical connection in which the intra-acrosomal calcium mobilization arm was subordinated to the fusion protein arm; somewhere after Rab3, the pathways became independent. CONCLUSIONS: We delineated the sequence of events that connect an external calcium signal to internal calcium mobilization during exocytosis. We have taken advantage of the versatility of the sperm model to investigate how cAMP, calcium, and the proteinaceous fusion machinery coordinate to accomplish secretion. Because the requirement of calcium from two different sources is not unique to sperm and fusion proteins are highly conserved, our findings might contribute to elucidate mechanisms that operate in regulated exocytosis in other secretory cell types.

GTP-bound Rab3A exhibits consecutive positive and negative roles during human sperm dense-core granule exocytosis.

Exocytosis of mammalian sperm dense-core secretory granule relies on the same fusion molecules as all other secretory cells; one such molecule is the small GTPase Rab3A. Here, we report an in-depth biochemical characterization of the role of Rab3A in secretion by scrutinizing the exocytotic response of streptolysin O-permeabilized human sperm to the acute application of a number of Rab3A-containing constructs and correlating the findings with those gathered with the endogenous protein. Full length, geranylgeranylated, and active Rab3A elicited human sperm exocytosis per se. With Rab3A/Rab22A chimeric proteins, we demonstrated that the carboxy-terminal domain of the Rab3A molecule was necessary and sufficient to promote exocytosis, whereas its amino-terminus prevented calcium-triggered secretion. Interestingly, full length Rab3A halted secretion when added after the docking of the acrosome to the plasma membrane. This effect depended on the inability of Rab3A to hydrolyze GTP. We combined modified immunofluorescence and acrosomal staining protocols to detect membrane fusion and the activation status of endogenous Rab3 simultaneously in individual cells, and found that GTP hydrolysis on endogenous Rab3 was mandatory for fusion pores to open. Our findings contribute to establishing that Rab3 modulates regulated exocytosis differently depending on the nucleotide bound and the exocytosis stage under study.

Munc18-1 controls SNARE protein complex assembly during human sperm acrosomal exocytosis.

The spermatozoon is a very specialized cell capable of carrying out a limited set of functions with high efficiency. Sperm are then excellent model cells to dissect fundamental processes such as regulated exocytosis. The secretion of the single dense-core granule of mammalian spermatozoa relies on the same highly conserved molecules and goes through the same stages as exocytosis in other types of cells. In this study, we describe the presence of Munc18-1 in human sperm and show that this protein has an essential role in acrosomal exocytosis. We observed that inactivation of endogenous Munc18-1 with a specific antibody precluded the stabilization of trans-SNARE complexes and inhibited acrosomal exocytosis. Addition of recombinant Munc18-1 blocked secretion by sequestering monomeric syntaxin, an effect that was rescued by α-soluble NSF attachment protein. By electron microscopy, we observed that both the anti-Munc18-1 antibody and recombinant Munc18-1 inhibited the docking of the acrosome to the plasma membrane. In conclusion, our results indicate that Munc18-1 plays a key role in the dynamics of trans-SNARE complex assembly and/or stabilization, a process that is necessary for the docking of the outer acrosomal membrane to the plasma membrane and subsequent fusion pore opening.

Rab27 and Rab3 sequentially regulate human sperm dense-core granule exocytosis.

Two so-called "secretory Rabs," Rab3 and Rab27, regulate late steps during dense-core vesicle exocytosis in neuroendocrine cells. Sperm contain a single large dense-core granule that is released by regulated exocytosis (termed the acrosome reaction) during fertilization or on exposure to inducers in vitro. Sperm exocytosis uses the same fusion machinery as neurons and neuroendocrine cells, with an additional requirement for active Rab3. Here we show that Rab27 is also required for the acrosome reaction, as demonstrated by the inability of inducers to elicit exocytosis when streptolysin O-permeabilized human sperm were loaded with inhibitory anti-Rab27 antibodies or the Rab27-GTP binding domain of the effector Slac2-b. The levels of GTP-bound Rab27 increased on initiation of exocytosis, as did the proportion of GTP-bound Rab3A. We have developed a fluorescence microscopy-based method for detecting endogenous Rab3A-GTP and Rab27-GTP in the acrosomal region of human sperm. Challenge with an inducer increased the population of cells exhibiting GTP-bound Rabs in this subcellular domain. Interestingly, introducing recombinant Rab27A loaded with GTP-γ-S into sperm elicited a remarkable increase in the number of cells evincing GTP-bound Rab3A. In the converse condition, recombinant Rab3A did not modify the percentage of Rab27-GTP-containing cells. Furthermore, Rab27A-GTP recruited a Rab3 GDP/GTP exchange factor (GEF) activity. Our findings suggest that Rab27/Rab3A constitutes a Rab-GEF cascade in dense-core vesicle exocytosis.

α-SNAP prevents docking of the acrosome during sperm exocytosis because it sequesters monomeric syntaxin.

α-SNAP has an essential role in membrane fusion that consists of bridging cis SNARE complexes to NSF. α-SNAP stimulates NSF, which releases itself, α-SNAP, and individual SNAREs that subsequently re-engage in the trans arrays indispensable for fusion. α-SNAP also binds monomeric syntaxin and NSF disengages the α-SNAP/syntaxin dimer. Here, we examine why recombinant α-SNAP blocks secretion in permeabilized human sperm despite the fact that the endogenous protein is essential for membrane fusion. The only mammalian organism with a genetically modified α-SNAP is the hyh mouse strain, which bears a M105I point mutation; males are subfertile due to defective sperm exocytosis. We report here that recombinant α-SNAP-M105I has greater affinity for the cytosolic portion of immunoprecipitated syntaxin than the wild type protein and in consequence NSF is less efficient in releasing the mutant. α-SNAP-M105I is a more potent sperm exocytosis blocker than the wild type and requires higher concentrations of NSF to rescue its effect. Unlike other fusion scenarios where SNAREs are subjected to an assembly/disassembly cycle, the fusion machinery in sperm is tuned so that SNAREs progress uni-directionally from a cis configuration in resting cells to monomeric and subsequently trans arrays in cells challenged with exocytosis inducers. By means of functional and indirect immunofluorescense assays, we show that recombinant α-SNAPs--wild type and M105I--inhibit exocytosis because they bind monomeric syntaxin and prevent this SNARE from assembling with its cognates in trans. Sequestration of free syntaxin impedes docking of the acrosome to the plasma membrane assessed by transmission electron microscopy. The N-terminal deletion mutant α-SNAP-(160-295), unable to bind syntaxin, affects neither docking nor secretion. The implications of this study are twofold: our findings explain the fertility defect of hyh mice and indicate that assembly of SNAREs in trans complexes is essential for docking.

Epac activates the small G proteins Rap1 and Rab3A to achieve 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 C-dependent 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.

Sperm from hyh mice carrying a point mutation in alphaSNAP have a defect in acrosome reaction.

Hydrocephalus with hop gait (hyh) is a recessive inheritable disease that arose spontaneously in a mouse strain. A missense mutation in the Napa gene that results in the substitution of a methionine for isoleucine at position 105 (M105I) of alphaSNAP has been detected in these animals. alphaSNAP is a ubiquitous protein that plays a key role in membrane fusion and exocytosis. In this study, we found that male hyh mice with a mild phenotype produced morphologically normal and motile sperm, but had a strongly reduced fertility. When stimulated with progesterone or A23187 (a calcium ionophore), sperm from these animals had a defective acrosome reaction. It has been reported that the M105I mutation affects the expression but not the function of the protein. Consistent with an hypomorphic phenotype, the testes and epididymides of hyh mice had low amounts of the mutated protein. In contrast, sperm had alphaSNAP levels indistinguishable from those found in wild type cells, suggesting that the mutated protein is not fully functional for acrosomal exocytosis. Corroborating this possibility, addition of recombinant wild type alphaSNAP rescued exocytosis in streptolysin O-permeabilized sperm, while the mutant protein was ineffective. Moreover, addition of recombinant alphaSNAP. M105I inhibited acrosomal exocytosis in permeabilized human and wild type mouse sperm. We conclude that the M105I mutation affects the expression and also the function of alphaSNAP, and that a fully functional alphaSNAP is necessary for acrosomal exocytosis, a key event in fertilization.

PTP1B dephosphorylates N-ethylmaleimide-sensitive factor and elicits SNARE complex disassembly during human sperm exocytosis.

The reversible phosphorylation of tyrosyl residues in proteins is a cornerstone of the signaling pathways that regulate numerous cellular responses. Protein tyrosine phosphorylation is controlled through the concerted actions of protein-tyrosine kinases and phosphatases. The goal of the present study was to unveil the mechanisms by which protein tyrosine dephosphorylation modulates secretion. The acrosome reaction, a specialized type of regulated exocytosis undergone by sperm, is initiated by calcium and carried out by a number of players, including tyrosine kinases and phosphatases, and fusion-related proteins such as Rab3A, alpha-SNAP, N-ethylmaleimide-sensitive factor (NSF), SNAREs, complexin, and synaptotagmin VI. We report here that inducers were unable to elicit the acrosome reaction when permeabilized human sperm were loaded with anti-PTP1B antibodies or with the dominant-negative mutant PTP1B D181A; subsequent introduction of wild type PTP1B or NSF rescued exocytosis. Wild type PTP1B, but not PTP1B D181A, caused cis SNARE complex dissociation during the acrosome reaction through a mechanism involving NSF. Unlike its non-phosphorylated counterpart, recombinant phospho-NSF failed to dissociate SNARE complexes from rat brain membranes. These results strengthen our previous observation that NSF activity is regulated rather than constitutive during sperm exocytosis and indicate that NSF must be dephosphorylated by PTP1B to disassemble SNARE complexes. Interestingly, phospho-NSF served as a substrate for PTP1B in an in vitro assay. Our findings demonstrate that phosphorylation of NSF on tyrosine residues prevents its SNARE complex dissociation activity and establish for the first time a role for PTP1B in the modulation of the membrane fusion machinery.

Complexin/synaptotagmin interplay controls acrosomal exocytosis.

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.

Acrosomal exocytosis, a special type of regulated secretion.

The 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 alphaSNAP 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.

Calcium-induced acrosomal exocytosis requires cAMP acting through a protein kinase A-independent, Epac-mediated pathway.

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. Ca2+ is the archetypical trigger of regulated exocytosis, and we show here that its effects on acrosomal release are fully mediated by cAMP. Ca2+ 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 dibutyryl-cAMP 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 Ca2+. 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 Ca2+, and ultimately, bilayer mixing and fusion. cAMP-elicited exocytosis was sensitive to anti-alpha-SNAP, anti-NSF, and anti-Rab3A antibodies, to intra-acrosomal Ca2+ 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 Ca2+ in exocytosis.

Dynamics of SNARE assembly and disassembly during sperm acrosomal exocytosis.

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/alpha-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/alpha-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.