The pair ceramide 1-phosphate/ceramide kinase regulates intracellular calcium and progesterone-induced human sperm acrosomal exocytosis.

Before fertilization, spermatozoa must undergo calcium-regulated acrosome exocytosis in response to physiological stimuli such as progesterone and zona pellucida. Our laboratory has elucidated the signaling cascades accomplished by different sphingolipids during human sperm acrosomal exocytosis. Recently, we established that ceramide increases intracellular calcium by activating various channels and stimulating the acrosome reaction. However, whether ceramide induces exocytosis on its own, activation of the ceramide kinase/ceramide 1-phosphate (CERK/C1P) pathway or both is still an unsolved issue. Here, we demonstrate that C1P addition induces exocytosis in intact, capacitated human sperm. Real-time imaging in single-cell and calcium measurements in sperm population showed that C1P needs extracellular calcium to induce [Ca2+]i increase. The sphingolipid triggered the cation influx through voltage-operated calcium (VOC) and store-operated calcium (SOC) channels. However, it requires calcium efflux from internal stores through inositol 3-phosphate receptors (IP3R) and ryanodine receptors (RyR) to achieve calcium rise and the acrosome reaction. We report the presence of the CERK in human spermatozoa, the enzyme that catalyzes C1P synthesis. Furthermore, CERK exhibited calcium-stimulated enzymatic activity during the acrosome reaction. Exocytosis assays using a CERK inhibitor demonstrated that ceramide induces acrosomal exocytosis, mainly due to C1P synthesis. Strikingly, progesterone required CERK activity to induce intracellular calcium increase and acrosome exocytosis. This is the first report, implicating the bioactive sphingolipid C1P in the physiological progesterone pathway leading to the sperm acrosome reaction.

Cortisol modulates Ca2+ signaling and acrosome reaction in human sperm.

BACKGROUND: Stress and elevated cortisol levels have negative effects on fertility, although there is controversy about the effect of cortisol on human sperm. One study reported that hydrocortisone (HC), the synthetic form of cortisol, does not activate CatSper channel but is able to inhibit its activation by progesterone (Pg). However, subsequent reports showed that HC has an agonist effect on CatSper, producing intracellular Ca2+ ([Ca2+ ]i ) increases. These studies were performed using only electrophysiological techniques and fluorometric measurements of Ca2+ . Therefore, it is important to evaluate the effects of different HC concentrations on human sperm physiology using other approaches and techniques. OBJECTIVE: To analyze the effects of different HC concentrations on human sperm physiology and to evaluate the association of perceived stress, anxiety and depression with acrosome reaction (AR), and semen quality. MATERIALS AND METHODS: In capacitated human sperm, we tested the effects of HC on [Ca2+ ]i mobilization and AR. Furthermore, the perceived stress and symptoms of anxiety and depression were evaluated with standardized psychometric tests. RESULTS: HC induces a low increase in [Ca2+ ]i compared to Pg response while, at higher concentrations, HC inhibits the Pg-induced Ca2+ response. Also, low concentrations of HC induce AR and when co-incubating it with Pg, the percentages of AR are decreased. Finally, the anxiety symptoms are negatively correlated with Pg induced AR and semen quality. DISCUSSION: HC has an agonist effect on [Ca2+ ]i mobilization and AR. However, our data also suggest that HC may compete for the binding site of Pg. Additionally, the association between anxiety and decreased AR could be explained by high cortisol levels. CONCLUSION: Our data showed that HC interferes with the Pg action. Therefore, the negative correlation between anxiety symptoms and Pg-induced AR is due to the accompanying high cortisol levels of this condition and its competitive action with Pg.

When left does not seem right: epigenetic and bioelectric differences between left- and right-sided breast cancer.

BACKGROUND: During embryogenesis lateral symmetry is broken, giving rise to Left/Right (L/R) breast tissues with distinct identity. L/R-sided breast tumors exhibit consistently-biased incidence, gene expression, and DNA methylation. We postulate that a differential L/R tumor-microenvironment crosstalk generates different tumorigenesis mechanisms. METHODS: We performed in-silico analyses on breast tumors of public datasets, developed xenografted tumors, and conditioned MDA-MB-231 cells with L/R mammary extracts. RESULTS: We found L/R differential DNA methylation involved in embryogenic and neuron-like functions. Focusing on ion-channels, we discovered significant L/R epigenetic and bioelectric differences. Specifically, L-sided cells presented increased methylation of hyperpolarizing ion channel genes and increased Ca2+ concentration and depolarized membrane potential, compared to R-ones. Functional consequences were associated with increased proliferation in left tumors, assessed by KI67 expression and mitotic count. CONCLUSIONS: Our findings reveal considerable L/R asymmetry in cancer processes, and suggest specific L/R epigenetic and bioelectric differences as future targets for cancer therapeutic approaches in the breast and many other paired organs.

Direct activation of the proton channel by albumin leads to human sperm capacitation and sustained release of inflammatory mediators by neutrophils.

Human voltage-gated proton channels (hHv1) extrude protons from cells to compensate for charge and osmotic imbalances due metabolism, normalizing intracellular pH and regulating protein function. Human albumin (Alb), present at various levels throughout the body, regulates oncotic pressure and transports ligands. Here, we report Alb is required to activate hHv1 in sperm and neutrophils. Dose-response studies reveal the concentration of Alb in semen is too low to activate hHv1 in sperm whereas the higher level in uterine fluid yields proton efflux, allowing capacitation, the acrosomal reaction, and oocyte fertilization. Likewise, Alb activation of hHv1 in neutrophils is required to sustain production and release of reactive oxygen species during the immune respiratory burst. One Alb binds to both voltage sensor domains (VSDs) in hHv1, enhancing open probability and increasing proton current. A computational model of the Alb-hHv1 complex, validated by experiments, identifies two sites in Alb domain II that interact with the VSDs, suggesting an electrostatic gating modification mechanism favoring the active "up" sensor conformation. This report shows how sperm are triggered to fertilize, resolving how hHv1 opens at negative membrane potentials in sperm, and describes a role for Alb in physiology that will operate in the many tissues expressing hHv1.

Epac activation induces an extracellular Ca2+ -independent Ca2+ wave that triggers acrosome reaction in human spermatozoa.

BACKGROUND: The signaling pathways of the intracellular second messengers cAMP and Ca2+ play a crucial role in numerous physiological processes in human spermatozoa. One such process is the acrosome reaction (AR), which is necessary for spermatozoa to traverse the egg envelope and to expose a fusogenic membrane allowing the egg-sperm fusion. Progesterone and zona pellucida elicit an intracellular Ca2+ increase that is needed for the AR in the mammalian spermatozoa. This increase is mediated by an initial Ca2+ influx but also by a release from intracellular Ca2+ stores. It is known that intracellular Ca2+ stores play a central role in the regulation of [Ca2+ ]i and in the generation of complex Ca2+ signals such as oscillations and waves. In the human spermatozoa, it has been proposed that the cAMP analog and specific agonist of Epac 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (2'-O-Me-cAMP) elicits an intracellular Ca2+ release involved in the AR. OBJECTIVE: To identify the molecular entities involved in the Ca2+ mobilization triggered by 2'-O-Me-cAMP in human spermatozoa. MATERIALS AND METHODS: In capacitated human spermatozoa, we monitored Ca2+ dynamics and the occurrence of the AR in real time using Fluo 3-AM and FM4-64 in a Ca2+ -free medium. RESULTS: Epac activation by 2'-O-Me-cAMP induced a Ca2+ wave that started in the midpiece and propagated to the acrosome region. This Ca2+ response was sensitive to rotenone, CGP, xestospongin, NED-19, and thapsigargin, suggesting the participation of different ion transporters (mitochondrial complex I and Na+ /Ca2+ exchanger, inositol 3-phosphate receptors, two-pore channels and internal store Ca2+ -ATPases). DISCUSSION: Our results suggest that Epac activation promotes a dynamic crosstalk between three different intracellular Ca2+ stores: the mitochondria, the redundant nuclear envelope, and the acrosome. CONCLUSION: The Ca2+ wave triggered by Epac activation is necessary to induce the AR and to enhance the flagellar beat.

Male Decapacitation Factor SPINK3 Blocks Membrane Hyperpolarization and Calcium Entry in Mouse Sperm.

Mammalian sperm acquire ability to fertilize through a process called capacitation, occurring after ejaculation and regulated by both female molecules and male decapacitation factors. Bicarbonate and calcium present in the female reproductive tract trigger capacitation in sperm, leading to acrosomal responsiveness and hyperactivated motility. Male decapacitating factors present in the semen avert premature capacitation, until detached from the sperm surface. However, their mechanism of action remains elusive. Here we describe for the first time the molecular basis for the decapacitating action of the seminal protein SPINK3 in mouse sperm. When present in the capacitating medium, SPINK3 inhibited Src kinase, a modulator of the potassium channel responsible for plasma membrane hyperpolarization. Lack of hyperpolarization affected calcium channels activity, impairing the acquisition of acrosomal responsiveness and blocking hyperactivation. Interestingly, SPINK3 acted only on non-capacitated sperm, as it did not bind to capacitated cells. Binding selectivity allows its decapacitating action only in non-capacitated sperm, without affecting capacitated cells.

Ceramide induces a multicomponent intracellular calcium increase triggering the acrosome secretion in human sperm.

Exocytosis of spermatozoon's secretory vesicle, named acrosome reaction (AR), is a regulated event that plays a central role in fertilization. It is coupled to a complex calcium signaling. Ceramide is a multitasking lipid involved in exocytosis. Nevertheless, its effect on secretion is controversial and the underlying cellular and molecular mechanisms remain unknown. Human spermatozoa are useful to dissect the role of ceramide in secretion given that the gamete is not capable to undergo any trafficking mechanisms other than exocytosis. We report for the first time, the presence of sphingolipid metabolism enzymes such as neutral-sphingomyelinase and ceramide synthase in sperm. Ceramidases are also present and active. Both the addition of cell-permeable ceramide and the rise of the endogenous one, increase intracellular calcium acting as potent inducers of exocytosis. Ceramide triggers AR in capacitated spermatozoa and enhances the gamete response to progesterone. The lipid induces physiological ultrastructural changes in the acrosome and triggers an exocytosis-signaling cascade involving protein tyrosine phosphatase 1B and VAMP2. Real-time imaging showed an increment of calcium in the cytosol upon ceramide treatment either in the absence or in the presence of extracellular calcium. Pharmacological experiments demonstrate that at early stages the process involves ryanodine receptors, CatSper (calcium channel of sperm), and store-operated calcium channels. We set out the signaling sequence of events that connect ceramide to internal calcium mobilization and external calcium signals during secretion. These results allow the coordination of lipids and proteins in a pathway that accomplishes secretion. Our findings contribute to the understanding of ceramide's role in regulated exocytosis and fertilization.

Role of human Hv1 channels in sperm capacitation and white blood cell respiratory burst established by a designed peptide inhibitor.

Using a de novo peptide inhibitor, Corza6 (C6), we demonstrate that the human voltage-gated proton channel (hHv1) is the main pathway for H+ efflux that allows capacitation in sperm and permits sustained reactive oxygen species (ROS) production in white blood cells (WBCs). C6 was identified by a phage-display strategy whereby ∼1 million novel peptides were fabricated on an inhibitor cysteine knot (ICK) scaffold and sorting on purified hHv1 protein. Two C6 peptides bind to each dimeric channel, one on the S3-S4 loop of each voltage sensor domain (VSD). Binding is cooperative with an equilibrium affinity (Kd) of ∼1 nM at -50 mV. As expected for a VSD-directed toxin, C6 inhibits by shifting hHv1 activation to more positive voltages, slowing opening and speeding closure, effects that diminish with membrane depolarization.

Live imaging of cortical granule exocytosis reveals that in vitro matured mouse oocytes are not fully competent to secrete their content.

Oocyte in vitro maturation does not entirely support all the nuclear and cytoplasmic changes that occur physiologically, and it is poorly understood whether in vitro maturation affects the competence of cortical granules to secrete their content during cortical reaction. Here, we characterize cortical granule exocytosis (CGE) in live mouse oocytes activated by strontium chloride using the fluorescent lectin FITC-LCA. We compared the kinetic of CGE between ovulated (in vivo matured, IVO) and in vitro matured (IVM) mouse oocytes. Results show that: (1) IVM oocytes have a severely reduced response to strontium chloride; (2) the low response was confirmed by quantification of remnant cortical granules in permeabilized cells and by a novel method to quantify the exudate in non-permeabilized cells; (3) the kinetic of CGE in IVO oocytes was rapid and synchronous; (4) the kinetic of CGE in IVM oocytes was delayed and asynchronous; (5) cortical granules in IVM oocytes show an irregular limit in regards to the cortical granule free domain. We propose the analysis of CGE in live oocytes as a biological test to evaluate the competence of IVM mouse oocytes.This article has an associated First Person interview with the first author of the paper.

ADP ribosylation factor 6 (ARF6) promotes acrosomal exocytosis by modulating lipid turnover and Rab3A activation.

Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. ARF6 (ADP-ribosylation factor 6) is a small GTPase implicated in exocytosis, but its downstream effectors remain elusive in this process. We combined biochemical, functional, and microscopy-based methods to show that ARF6 is present in human sperm, localizes to the acrosomal region, and is required for calcium and diacylglycerol-induced exocytosis. Results from pulldown assays show that ARF6 exchanges GDP for GTP in sperm challenged with different exocytic stimuli. Myristoylated and guanosine 5'-3-O-(thio)triphosphate (GTPγS)-loaded ARF6 (active form) added to permeabilized sperm induces acrosome exocytosis even in the absence of extracellular calcium. We explore the ARF6 signaling cascade that promotes secretion. We demonstrate that ARF6 stimulates a sperm phospholipase D activity to produce phosphatidic acid and boosts the synthesis of phosphatidylinositol 4,5-bisphosphate. We present direct evidence showing that active ARF6 increases phospholipase C activity, causing phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate-dependent intra-acrosomal calcium release. We show that active ARF6 increases the exchange of GDP for GTP on Rab3A, a prerequisite for secretion. We propose that exocytic stimuli activate ARF6, which is required for acrosomal calcium efflux and the assembly of the membrane fusion machinery. This report highlights the physiological importance of ARF6 as a key factor for human sperm exocytosis and fertilization.

Perfringolysin O as a useful tool to study human sperm physiology.

OBJECTIVE: To evaluate perfringolysin O, a cholesterol-dependent pore-forming cytolysin, as a tool to study several aspects of human sperm physiology. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): Human semen samples with normal parameters obtained from healthy donors. INTERVENTION(S): Interaction of recombinant perfringolysin O with human spermatozoa. MAIN OUTCOME MEASURE(S): Assessment of perfringolysin O binding to spermatozoa, tests for acrosome and plasma membrane integrity, and acrosomal exocytosis assays. RESULT(S): Perfringolysin O associated with human spermatozoa at 4°C. The binding was sensitive to changes in cholesterol concentrations and distribution occurring in the plasma membrane of these cells during capacitation. When perfringolysin O-treated sperm were incubated at 37°C, the plasma membrane became permeable, whereas the acrosome membrane remained intact. Permeabilized spermatozoa were able to respond to exocytic stimuli. The process was inhibited by proteins that interfere with membrane fusion, indicating that large molecules, including antibodies, were able to permeate into the spermatozoa. CONCLUSION(S): PFO is a useful probe to assess changes in the amount and distribution of the active sterol fraction present in the sperm plasma membrane. The toxin can be used for the efficient and selective permeabilization of this membrane, rendering a flexible experimental model suitable for studying molecular processes occurring in the sperm cytoplasm.

Diacylglycerol stimulates acrosomal exocytosis by feeding into a PKC- and PLD1-dependent positive loop that continuously supplies phosphatidylinositol 4,5-bisphosphate.

Acrosomal exocytosis involves a massive fusion between the outer acrosomal and the plasma membranes of the spermatozoon triggered by stimuli that open calcium channels at the plasma membrane. Diacylglycerol has been implicated in the activation of these calcium channels. Here we report that this lipid promotes the efflux of intraacrosomal calcium and triggers exocytosis in permeabilized human sperm, implying that diacylglycerol activates events downstream of the opening of plasma membrane channels. Furthermore, we show that calcium and diacylglycerol converge in a signaling pathway leading to the production of phosphatidylinositol 4,5-bisphosphate (PIP(2)). Addition of diacylglycerol promotes the PKC-dependent activation of PLD1. Rescue experiments adding phosphatidic acid or PIP(2) and direct measurement of lipid production suggest that both PKC and PLD1 promote PIP(2) synthesis. Inhibition of different steps of the pathway was reverted by adenophostin, an agonist of IP(3)-sensitive calcium channels, indicating that PIP(2) is necessary to keep these channels opened. However, phosphatidic acid, PIP(2), or adenophostin could not trigger exocytosis by themselves, indicating that diacylglycerol must also activate another factor. We found that diacylglycerol and phorbol ester stimulate the accumulation of the GTP-bound form of Rab3A. Together our results indicate that diacylglycerol promotes acrosomal exocytosis by i) maintaining high levels of IP(3) - an effect that depends on a positive feedback loop leading to the production of PIP(2) - and ii) stimulating the activation of Rab3A, which in turn initiates a cascade of protein interactions leading to the assembly of SNARE complexes and membrane fusion.

TRPM8 in mouse sperm detects temperature changes and may influence the acrosome reaction.

Changes in the concentration of intracellular Ca(2+) ([Ca(2+) ]i) trigger and/or regulate principal sperm functions during fertilization, such as motility, capacitation, and the acrosome reaction (AR). Members of the large TRP channel family participate in a variety of Ca(2+) -dependent cell signaling processes. The eight TRPM channel members constitute one of the seven groups belonging to this family. Here we document using RT-PCR experiments the presence of Trpm2, 4, 7, and 8 in mouse spermatogenic cells. Trpm8 transcription is up-regulated after day 30. The localization of TRPM8 protein in mouse sperm was confirmed by immunocytochemistry and Western blots. Patch clamp recordings in testicular mouse sperm revealed TRPM8 agonist (menthol and icilin) activated currents sensitive to TRPM8 inhibitors N-(4-t-Butylphenyl)-4-(3-Chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide (BCTC) and capsazepine. These findings are consistent with the presence of functional TRPM8 in mouse sperm. Furthermore, menthol induced a [Ca(2+) ]i increase and the AR in these cells, that were inhibited by capsazepine (20 µM) and BCTC (1.6 µM). Notably, the progesterone and zona pellucida-induced AR was significantly (>40%) inhibited by BCTC and capsazepine, suggesting the possible participation of TRPM8 channels in this reaction. TRPM family members present in sperm could be involved in other important signaling events, such as thermotaxis, chemotaxis, and mechanosensory transduction.

The opening of maitotoxin-sensitive calcium channels induces the acrosome reaction in human spermatozoa: differences from the zona pellucida.

The acrosome reaction (AR), an absolute requirement for spermatozoa and egg fusion, requires the influx of Ca²(+) into the spermatozoa through voltage-dependent Ca²(+) channels and store-operated channels. Maitotoxin (MTx), a Ca²(+)-mobilizing agent, has been shown to be a potent inducer of the mouse sperm AR, with a pharmacology similar to that of the zona pellucida (ZP), possibly suggesting a common pathway for both inducers. Using recombinant human ZP3 (rhZP3), mouse ZP and two MTx channel blockers (U73122 and U73343), we investigated and compared the MTx- and ZP-induced ARs in human and mouse spermatozoa. Herein, we report that MTx induced AR and elevated intracellular Ca²(+) ([Ca²(+)](i)) in human spermatozoa, both of which were blocked by U73122 and U73343. These two compounds also inhibited the MTx-induced AR in mouse spermatozoa. In disagreement with our previous proposal, the AR triggered by rhZP3 or mouse ZP was not blocked by U73343, indicating that in human and mouse spermatozoa, the AR induction by the physiological ligands or by MTx occurred through distinct pathways. U73122, but not U73343 (inactive analogue), can block phospholipase C (PLC). Another PLC inhibitor, edelfosine, also blocked the rhZP3- and ZP-induced ARs. These findings confirmed the participation of a PLC-dependent signalling pathway in human and mouse zona protein-induced AR. Notably, edelfosine also inhibited the MTx-induced mouse sperm AR but not that of the human, suggesting that toxin-induced AR is PLC-dependent in mice and PLC-independent in humans.

Sphingosine 1-phosphate and sphingosine kinase are involved in a novel signaling pathway leading to acrosomal exocytosis.

Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. Sphingosine 1-phosphate is a bioactive sphingolipid that regulates crucial physiological processes. Here we report that this lipid triggers acrosomal exocytosis in human sperm by a mechanism involving a G(i)-coupled receptor. Real-time imaging showed a remarkable increase of cytosolic calcium upon activation with sphingosine 1-phosphate and pharmacological experiments indicate that the process requires extracellular calcium influx through voltage and store-operated calcium channels and efflux from intracellular stores through inositol 1,4,5-trisphosphate-sensitive calcium channels. Sphingosine 1-phosphate-induced exocytosis requires phospholipase C and protein kinase C activation. We investigated possible sources of the lipid. Western blot indicates that sphingosine kinase 1 is present in spermatozoa. Indirect immunofluorescence showed that phorbol ester, a potent protein kinase C activator that can also trigger acrosomal exocytosis, redistributes sphingosine kinase 1 to the acrosomal region. Functional assays showed that phorbol ester-induced exocytosis depends on the activation of sphingosine kinase 1. Furthermore, incorporation of (32)P to sphingosine demonstrates that cells treated with the phorbol ester increase their sphingosine kinase activity that yields sphingosine 1-phosphate. We present here the first evidence indicating that human spermatozoa produce sphingosine 1-phosphate when challenged with an exocytic stimulus. These observations point to a new role of sphingosine 1-phosphate in a signaling cascade that facilitates acrosome reaction providing some clues about novel lipid molecules involved in exocytosis.

TRPM8, a versatile channel in human sperm.

BACKGROUND: The transient receptor potential channel (TRP) family includes more than 30 proteins; they participate in various Ca(2+) dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca(2+) ([Ca(2+)]i). Ca(2+) triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored. PRINCIPAL FINDINGS: Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature) and antagonists (BCTC and capsazepine). Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 microM) and 80% by BCTC (1.6 microM). Activation of TRPM8 either by temperature or menthol induced [Ca(2+)]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 microM) and BCTC (1.6 microM). However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway. CONCLUSIONS: This is the first report dealing with the presence of a thermo sensitive channel (TRPM8) in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis.

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.

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.