Ca2+ Signals Generated by CatSper and Ca2+ Stores Regulate Different Behaviors in Human Sperm

[Ca²⁺]_i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca²⁺ signaling pathway used. Activation of CatSper (by raising pH_i or stimulating with progesterone) caused sustained [Ca²⁺]_i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 μm thimerosal to mobilize stored Ca²⁺ caused sustained [Ca²⁺]_i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pH_i and mobilized Ca²⁺ stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca²⁺-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca²⁺]_i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca²⁺ at the sperm neck can be mobilized by Ca²⁺-induced Ca²⁺ release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca²⁺ store, which may be regulated by capacitation and NO from the cumulus.     

The CatSper channel controls chemosensation in sea urchin sperm

Sperm guidance is controlled by chemical and physical cues. In many species, Ca²⁺ bursts in the flagellum govern navigation to the egg. In Arbacia punctulata, a model system of sperm chemotaxis, a cGMP signaling pathway controls these Ca²⁺ bursts. The underlying Ca²⁺ channel and its mechanisms of activation are unknown. Here, we identify CatSper Ca²⁺ channels in the flagellum of A. punctulata sperm. We show that CatSper mediates the chemoattractant-evoked Ca²⁺ influx and controls chemotactic steering; a concomitant alkalization serves as a highly cooperative mechanism that enables CatSper to transduce periodic voltage changes into Ca²⁺ bursts. Our results reveal intriguing phylogenetic commonalities but also variations between marine invertebrates and mammals regarding the function and control of CatSper. The variations probably reflect functional and mechanistic adaptations that evolved during the transition from external to internal fertilization.     

Identification of human and mouse CatSper3 and CatSper4 genes: Characterisation of a common interaction domain and evidence for expression in testis

Background  

CatSper1 and CatSper2 are two recently identified channel-like proteins, which show sperm specific expression patterns. Through targeted mutagenesis in the mouse, CatSper1 has been shown to be required for fertility, sperm motility and for cAMP induced Ca²⁺ current in sperm. Both channels resemble a single pore forming repeat from a four repeat voltage dependent Ca²⁺ /Na⁺ channel. However, neither CatSper1 or CatSper2 have been shown to function as cation channels when transfected into cells, singly or in conjunction. As the pore forming units of voltage gated cation channels form a tetramer it has been suggested that the known CatSper proteins require additional subunits and/or interaction partners to function.     

A Model of CatSper Channel Mediated Calcium Dynamics in Mammalian Spermatozoa

CatSpers are calcium (Ca²⁺) channels that are located along the principal piece of mammalian sperm flagella and are directly linked to sperm motility and hyperactivation. It has been observed that Ca²⁺ entry through CatSper channels triggers a tail to head Ca²⁺ propagation in mouse sperm, as well as a sustained increase of Ca²⁺ in the head. Here, we develop a mathematical model to investigate this propagation and sustained increase in the head. A 1-d reaction-diffusion model tracking intracellular Ca²⁺ with flux terms for the CatSper channels, a leak flux, and plasma membrane Ca²⁺ clearance mechanism is studied. Results of this simple model exhibit tail to head Ca²⁺ propagation, but no sustained increase in the head. Therefore, in this model, a simple plasma membrane pump-leak system with diffusion in the cytosol cannot account for these experimentally observed results. It has been proposed that Ca²⁺ influx from the CatSper channels induce additional Ca²⁺ release from an internal store. We test this hypothesis by examining the possible role of Ca²⁺ release from the redundant nuclear envelope (RNE), an inositol 1,4,5-trisphosphate (IP₃) gated Ca²⁺ store in the neck. The simple model is extended to include an equation for IP₃ synthesis, degradation, and diffusion, as well as flux terms for Ca²⁺ in the RNE. When IP₃ and the RNE are accounted for, the results of the model exhibit a tail to head Ca²⁺ propagation as well as a sustained increase of Ca²⁺ in the head.     

Pharmacological Targeting of Native CatSper Channels Reveals a Required Role in Maintenance of Sperm Hyperactivation

The four sperm-specific CatSper ion channel proteins are required for hyperactivated motility and male fertility, and for Ca²⁺ entry evoked by alkaline depolarization. In the absence of external Ca²⁺, Na⁺ carries current through CatSper channels in voltage-clamped sperm. Here we show that CatSper channel activity can be monitored optically with the [Na⁺]_i-reporting probe SBFI in populations of intact sperm. Removal of external Ca²⁺ increases SBFI signals in wild-type but not CatSper2-null sperm. The rate of the indicated rise of [Na⁺]_i is greater for sperm alkalinized with NH₄Cl than for sperm acidified with propionic acid, reflecting the alkaline-promoted signature property of CatSper currents. In contrast, the [Na⁺]_i rise is slowed by candidate CatSper blocker HC-056456 (IC₅₀ ∼3 µM). HC-056456 similarly slows the rise of [Ca²⁺]_i that is evoked by alkaline depolarization and reported by fura-2. HC-056456 also selectively and reversibly decreased CatSper currents recorded from patch-clamped sperm. HC-056456 does not prevent activation of motility by HCO₃ ⁻ but does prevent the development of hyperactivated motility by capacitating incubations, thus producing a phenocopy of the CatSper-null sperm. When applied to hyperactivated sperm, HC-056456 causes a rapid, reversible loss of flagellar waveform asymmetry, similar to the loss that occurs when Ca²⁺ entry through the CatSper channel is terminated by removal of external Ca²⁺. Thus, open CatSper channels and entry of external Ca²⁺ through them sustains hyperactivated motility. These results indicate that pharmacological targeting of the CatSper channel may impose a selective late-stage block to fertility, and that high-throughput screening with an optical reporter of CatSper channel activity may identify additional selective blockers with potential for male-directed contraception.     

CRISP1 as a novel CatSper regulator that modulates sperm motility and orientation during fertilization

Ca²⁺-dependent mechanisms are critical for successful completion of fertilization. Here, we demonstrate that CRISP1, a sperm protein involved in mammalian fertilization, is also present in the female gamete and capable of modulating key sperm Ca²⁺ channels. Specifically, we show that CRISP1 is expressed by the cumulus cells that surround the egg and that fertilization of cumulus–oocyte complexes from CRISP1 knockout females is impaired because of a failure of sperm to penetrate the cumulus. We provide evidence that CRISP1 stimulates sperm orientation by modulating sperm hyperactivation, a vigorous motility required for penetration of the egg vestments. Moreover, patch clamping of sperm revealed that CRISP1 has the ability to regulate CatSper, the principal sperm Ca²⁺ channel involved in hyperactivation and essential for fertility. Given the critical role of Ca²⁺ for sperm motility, we propose a novel CRISP1-mediated fine-tuning mechanism to regulate sperm hyperactivation and orientation for successful penetration of the cumulus during fertilization.     

Conservation of the Ca2+-permeability through the voltage sensor domain of mammalian CatSper subunit

Cation channel of Spermatozoa (CatSper) is one of the voltage-gated ion channels consisting of voltage sensor domains (VSDs) and pore-gate domains. CatSper is exclusively expressed in spermatozoa and indispensable for Ca²⁺ influx into cytosol. Recently, we have reported that the VSD of ascidian CatSper induces Ca²⁺-permeable pathways in heterologous expression systems. However, it is not known whether ion permeability through the VSD of CatSper is conserved in mammals. In the present study, electrophysiology and fluorometry in Xenopus oocytes revealed that Ca²⁺-permeable paths are also formed by expressing the VSD of murine CatSper. We also examined the permeability to monovalent cations other than Na⁺ in the VSD of ascidian CatSper.     

Coupling biochemistry and hydrodynamics captures hyperactivated sperm motility in a simple flagellar model

Hyperactivation in mammalian sperm is characterized by highly asymmetrical waveforms and an increase in the amplitude of flagellar bends. It is important for the sperm to be able to achieve hyperactivated motility in order to reach and fertilize the egg. Calcium (Ca²⁺) dynamics are known to play a large role in the initiation and maintenance of hyperactivated motility. Here we present an integrative model that couples the CatSper channel mediated Ca²⁺ dynamics of hyperactivation to a mechanical model of an idealized sperm flagellum in a 3-d viscous, incompressible fluid. The mechanical forces are due to passive stiffness properties and active bending moments that are a function of the local Ca²⁺ concentration along the length of the flagellum. By including an asymmetry in bending moments to reflect an asymmetry in the axoneme's response to Ca²⁺, we capture the transition from activated motility to hyperactivated motility. We examine the effects of elastic properties of the flagellum and the Ca²⁺ dynamics on the overall swimming patterns. The swimming velocities of the model flagellum compare well with data for hyperactivated mouse sperm.     

Tyrosine phosphorylation signaling regulates Ca2+ entry by affecting intracellular pH during human sperm capacitation

Capacitation is a mandatory process for the acquisition of mammalian sperm fertilization competence and involves the activation of a complex and still not fully understood system of signaling pathways. Under in vitro conditions, there is an increase in both protein tyrosine phosphorylation (pTyr) and intracellular Ca²⁺ levels in several species. In human sperm, results from our group revealed that pTyr signaling can be blocked by inhibiting proline-rich tyrosine kinase 2 (PYK2). Based on the role of PYK2 in other cell types, we investigated whether the PYK2-dependent pTyr cascade serves as a sensor for Ca ²⁺ signaling during human sperm capacitation. Flow cytometry studies showed that exposure of sperm to the PYK2 inhibitor N-[2-[[[2-[(2,3-dihydro-2-oxo-1 H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]- N-methyl-methanesulfonamide hydrate (PF431396) produced a significant and concentration-dependent reduction in intracellular Ca ²⁺ levels during capacitation. Further studies revealed that PF431396-treated sperm exhibited a decrease in the activity of CatSper, a key sperm Ca ²⁺ channel. In addition, time course studies during capacitation in the presence of PF431396 showed a significant and sustained decrease in both intracellular Ca ²⁺ and pH levels after 2 hr of incubation, temporarily coincident with the activation of PYK2 during capacitation. Interestingly, decreases in Ca ²⁺ levels and progressive motility caused by PF431396 were reverted by inducing intracellular alkalinization with NH ₄Cl, without affecting the pTyr blockage. Altogether, these observations support pTyr as an intracellular sensor for Ca ²⁺ entry in human sperm through regulation of cytoplasmic pH. These results contribute to a better understanding of the modulation of the polymodal CatSper and signaling pathways involved in human sperm capacitation.     

Signaling pathways involved in human sperm hyperactivated motility stimulated by Zn2+

To fertilize the egg, sperm cells must reside in the female reproductive tract for several hours during which they undergo chemical and motility changes collectively called capacitation. During capacitation, the sperm develop a unique type of motility known as hyperactivated motility (HAM). The semen contains Zn²⁺ in millimolar concentrations, whereas in the female reproductive tract, the concentration is around 1 µM. In this study, we characterize the role of Zn²⁺ in human sperm capacitation focusing on its effect on HAM. Western blot analysis revealed the presence of GPR39‐type Zn‐receptor localized mainly in the sperm tail. Zn²⁺ at micromolar concentration stimulates HAM, which is mediated by a cascade involving GPR39–adenylyl cyclase (AC)–cyclic AMP (cAMP)–protein kinase A–tyrosine kinase Src (Src)–epidermal growth factor receptor and phospholipase C. Both the transmembrane AC and the soluble‐AC are involved in the stimulation of HAM by Zn²⁺. The development of HAM is precisely regulated by cAMP, in which relatively low concentration (5–10 µM) stimulated HAM, whereas at 30 µM no stimulation occurred. A similar response was seen when different concentrations of Zn²⁺ were added to the cells; low Zn²⁺ stimulated HAM, whereas at relatively high Zn²⁺, no effect was seen. We further demonstrate that the Ca²⁺‐channel CatSper involved in Zn²⁺‐stimulated HAM. These data support a role for extracellular Zn²⁺ acting via GPR39 to regulate signaling pathways in sperm capacitation, leading to HAM induction.     

Evolutionary Genomics Reveals Lineage-Specific Gene Loss and Rapid Evolution of a Sperm-Specific Ion Channel Complex: CatSpers and CatSperβ

The mammalian CatSper ion channel family consists of four sperm-specific voltage-gated Ca²⁺ channels that are crucial for sperm hyperactivation and male fertility. All four CatSper subunits are believed to assemble into a heteromultimeric channel complex, together with an auxiliary subunit, CatSperβ. Here, we report a comprehensive comparative genomics study and evolutionary analysis of CatSpers and CatSperβ, with important correlation to physiological significance of molecular evolution of the CatSper channel complex. The development of the CatSper channel complex with four CatSpers and CatSperβ originated as early as primitive metazoans such as the Cnidarian Nematostella vectensis. Comparative genomics revealed extensive lineage-specific gene loss of all four CatSpers and CatSperβ through metazoan evolution, especially in vertebrates. The CatSper channel complex underwent rapid evolution and functional divergence, while distinct evolutionary constraints appear to have acted on different domains and specific sites of the four CatSper genes. These results reveal unique evolutionary characteristics of sperm-specific Ca²⁺ channels and their adaptation to sperm biology through metazoan evolution.     

Signaling pathways involved in human sperm hyperactivated motility stimulated by Zn2+

To fertilize the egg, sperm cells must reside in the female reproductive tract for several hours during which they undergo chemical and motility changes collectively called capacitation. During capacitation, the sperm develop a unique type of motility known as hyperactivated motility (HAM). The semen contains Zn²⁺ in millimolar concentrations, whereas in the female reproductive tract the concentration is around 1 µM. In this study, we characterize the role of Zn ²⁺ in human sperm capacitation focusing on its effect on HAM. Western blot analysis revealed the presence of G protein‐coupled receptor 39 (GPR39) type Zn‐receptor localized mainly in the sperm tail. Zn ²⁺ at micromolar concentration stimulates HAM, which is mediated by a cascade involving GPR39‐AC‐cAMP‐PKA‐Src‐EGFR and phospholipase C. Both the transmembrane adenylyl cyclase (AC) and the soluble‐AC are involved in the stimulation of HAM by Zn ²⁺. The development of HAM is precisely regulated by cyclic adenosine monophosphate, in which relatively low concentration (5–10 µM) stimulated HAM, whereas at 30 µM no stimulation occurred. A similar response was seen when different concentrations of Zn ²⁺ were added to the cells; low Zn ²⁺ stimulated HAM, whereas at relatively high Zn ²⁺, no effect was seen. We further demonstrate that the Ca ²⁺‐channel CatSper involved in Zn ²⁺‐stimulated HAM. These data support a role for extracellular Zn ²⁺ acting via GPR39 to regulate signaling pathways in sperm capacitation, leading to HAM induction.     

An influx of extracelluar calcium is required for initiation of the human sperm acrosome reaction induced by human follicular fluid

The role of Ca²⁺ in the human sperm acrosome reaction was investigated using the fluorescent calcium indicator fura-2. Previous experiments have shown that a Sephadex G-75 column fraction of human follicular fluid can stimulate the human sperm acrosome reaction [Suarez SS, Wolf DP, Meizel S (1986): Gamete Res 14:107–121]. Using fura-2, we demonstrated that this Sephadex G-75 fraction also stimulates a rapid, transient increase in intracellular free Ca²⁺. This Ca²⁺ transient is blocked either by chelation of extracellular calcium or by addition of the Ca²⁺ antagonist La³⁺. We have also been able to stimulate the acrosome reaction in human sperm without significant loss of motility, using the divalent cation ionophore ionomycin. Acrosome reactions stimulated by whole follicular fluid, the G-75 fraction, or ionomycin are all blocked by removal of extracellular Ca²⁺. These results strongly suggest that an influx of extracellular Ca²⁺ is responsible for intiating the acrosome reaction in human sperm treated with human follicular fluid. This is the first demonstration in mammalian sperm that a potentially physiological stimulus can cause an increase in intracellular Ca²⁺ concomitant with the acrosome reaction.     

Plasma membrane calcium ATPase 4 (PMCA4) co‐ordinates calcium and nitric oxide signaling in regulating murine sperm functional activity

Reduced sperm motility (asthenospermia) and resulting infertility arise from deletion of the Plasma Membrane Ca²⁺‐ATPase 4 (Pmca4) gene which encodes the highly conserved Ca²⁺ efflux pump, PMCA4. This is the major Ca²⁺ clearance protein in murine sperm. Since the mechanism underlying asthenospermia in PMCA4's absence or reduced activity is unknown, we investigated if sperm PMCA4 negatively regulates nitric oxide synthases (NOSs) and when absent NO, peroxynitrite, and oxidative stress levels are increased. Using co‐immunoprecipitation (Co‐IP) and Fluorescence Resonance Energy Transfer (FRET), we show an association of PMCA4 with the NOSs in elevated cytosolic [Ca²⁺] in capacitated and Ca²⁺ ionophore‐treated sperm and with neuronal (nNOS) at basal [Ca²⁺] (ucapacitated sperm). FRET efficiencies for PMCA4‐eNOS were 35% and 23% in capacitated and uncapacitated sperm, significantly (p < 0.01) different, with the molecules being <10 nm apart. For PMCA4‐nNOS, this interaction was seen only for capacitated sperm where FRET efficiency was 24%, significantly (p < 0.05) higher than in uncapacitated sperm (6%). PMCA4 and the NOSs were identified as interacting partners in a quaternary complex that includes Caveolin1, which co‐immunoprecipitated with eNOS in a Ca²⁺‐dependent manner. In Pmca4^?/? sperm NOS activity was elevated twofold in capacitated/uncapacitated sperm (vs. wild‐type), accompanied by a twofold increase in peroxynitrite levels and significantly (p < 0.001) increased numbers of apoptotic germ cells. The data support a quaternary complex model in which PMCA4 co‐ordinates Ca²⁺ and NO signaling to maintain motility, with increased NO levels resulting in asthenospermia in Pmca4^?/? males. They suggest the involvement of PMCA4 mutations in human asthenospermia, with diagnostic relevance.     

A novel neuronal calcium sensor family protein, calaxin, is a potential Ca(2+)-dependent regulator for the outer arm dynein of metazoan cilia and flagella

Background information. Spermatozoa show several changes in flagellar waveform, such as upon fertilization. Ca²⁺ has been shown to play critical roles in modulating the waveforms of sperm flagella. However, a Ca²⁺‐binding protein in sperm flagella that regulates axonemal dyneins has not been fully characterized. Results. We identified a novel neuronal calcium sensor family protein, named calaxin (Ca²⁺‐binding axonemal protein), in sperm flagella of the ascidian Ciona intestinalis. Calaxin has three EF‐hand Ca²⁺‐binding motifs, and its orthologues are present in metazoan species, but not in yeast, green algae or plant. Immunolocalization revealed that calaxin is localized near the outer arm of the sperm flagellar axonemes. Moreover, it is distributed in adult tissues bearing epithelial cilia. An in vitro binding experiment indicated that calaxin binds to outer arm dynein. A cross‐linking experiment showed that calaxin binds to β‐tubulin in situ. Overlay experiments further indicated that calaxin binds the β‐dynein heavy chain of outer arm dynein in the presence of Ca²⁺. Conclusions. These results suggest that calaxin is a potential Ca²⁺‐dependent modulator of outer arm dynein in metazoan cilia and flagella.     

RIM‐binding proteins recruit BK‐channels to presynaptic release sites adjacent to voltage‐gated Ca2+‐channels

The active zone of presynaptic nerve terminals organizes the neurotransmitter release machinery, thereby enabling fast Ca²⁺‐triggered synaptic vesicle exocytosis. BK‐channels are Ca²⁺‐activated large‐conductance K⁺‐channels that require close proximity to Ca²⁺‐channels for activation and control Ca²⁺‐triggered neurotransmitter release by accelerating membrane repolarization during action potential firing. How BK‐channels are recruited to presynaptic Ca²⁺‐channels, however, is unknown. Here, we show that RBPs (for RIM‐binding proteins), which are evolutionarily conserved active zone proteins containing SH3‐ and FN3‐domains, directly bind to BK‐channels. We find that RBPs interact with RIMs and Ca²⁺‐channels via their SH3‐domains, but to BK‐channels via their FN3‐domains. Deletion of RBPs in calyx of Held synapses decreased and decelerated presynaptic BK‐currents and depleted BK‐channels from active zones. Our data suggest that RBPs recruit BK‐channels into a RIM‐based macromolecular active zone complex that includes Ca²⁺‐channels, synaptic vesicles, and the membrane fusion machinery, thereby enabling tight spatio‐temporal coupling of Ca²⁺‐influx to Ca²⁺‐triggered neurotransmitter release in a presynaptic terminal.     

Regulation of diacylglycerol production and protein kinase C stimulation during sperm- and PLCzeta-mediated mouse egg activation

Background information. At fertilization in mammalian eggs, the sperm induces a series of Ca²⁺ oscillations via the production of inositol 1,4,5‐trisphosphate. Increased inositol 1,4,5‐trisphosphate production appears to be triggered by a sperm‐derived PLCζ (phospholipase C‐ζ) that enters the egg after gamete fusion. The specific phosphatidylinositol 4,5‐bisphosphate hydrolytic activity of PLCζ implies that DAG (diacylglycerol) production, and hence PKC (protein kinase C) stimulation, also occurs during mammalian egg fertilization. Fertilization‐mediated increase in PKC activity has been demonstrated; however, its precise role is unclear. Results. We investigated PLCζ‐ and fertilization‐mediated generation of DAG in mouse eggs by monitoring plasma‐membrane translocation of a fluorescent DAG‐specific reporter. Consistent plasma‐membrane DAG formation at fertilization, or after injection of physiological concentrations of PLCζ, was barely detectable. However, when PLCζ is overexpressed in eggs, significant plasma‐membrane DAG production occurs in concert with a series of unexpected secondary high‐frequency Ca²⁺ oscillations. We show that these secondary Ca²⁺ oscillations can be mimicked in a variety of situations by the stimulation of PKC and that they can be prevented by PKC inhibition. The way PKC leads to secondary Ca²⁺ oscillations appears to involve Ca²⁺ influx and the loading of thapsigargin‐sensitive Ca²⁺ stores. Conclusions. Our results suggest that overproduction of DAG in PLCζ‐injected eggs can lead to PKC‐mediated Ca²⁺ influx and subsequent overloading of Ca²⁺ stores. These results suggest that DAG generation in the plasma membrane of fertilizing mouse eggs is minimized since it can perturb egg Ca²⁺ homoeostasis via excessive Ca²⁺ influx.     

Distinct Ca2+ channels maintain a high motility state of the sperm that may be needed for penetration of egg jelly of the newt,Cynops pyrrhogaster

Activation state of sperm motility named “hyperactivation” enables mammalian sperm to progress through the oviductal matrix, although a similar state of sperm motility is unknown in non‐mammalian vertebrates at fertilization. Here, we found a high motility state of the sperm in the newt Cynops pyrrhogaster. It was predominantly caused in egg jelly extract (JE) and characterized by a high wave velocity of the undulating membrane (UM) that was significantly higher at the posterior midpiece. An insemination assay suggested that the high motility state might be needed for sperm to penetrate the egg jelly, which is the accumulated oviductal matrix. Specific characteristics of the high motility state were completely abrogated by a high concentration of verapamil, which blocks the L‐type and T‐type voltage‐dependent Ca²⁺ channels (VDCCs). Mibefradil, a dominant blocker of T‐type VDCCs, suppressed the wave of the UM at the posterior midpiece with separate wave propagation from both the anterior midpiece and the posterior principal piece. In addition, nitrendipine, a dominant L‐type VDCC blocker, weakened the wave of the UM, especially in the anterior midpiece. Live Ca²⁺ imaging showed that, compared with the intact sperm in the JE, the relative intracellular Ca²⁺ level changed especially in the anterior and posterior ends of the midpiece of the blocker‐treated sperm. These suggest that different types of Ca²⁺ channels mediate the intracellular Ca²⁺ level predominantly in the anterior and posterior ends of the midpiece to maintain the high motility state of the newt sperm.     

Characterization of NAADP-mediated calcium signaling in human spermatozoa

Ca²⁺ signaling in spermatozoa plays a crucial role during processes such as capacitation and release of the acrosome, but the underlying molecular mechanisms still remain unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca²⁺-releasing second messenger in a variety of cellular processes. The presence of a NAADP synthesizing enzyme in sea urchin sperm has been previously reported, suggesting a possible role of NAADP in sperm Ca²⁺ signaling. In this work we used in vitro enzyme assays to show the presence of a novel NAADP synthesizing enzyme in human sperm, and to characterize its sensitivity to Ca²⁺ and pH. Ca²⁺ fluorescence imaging studies demonstrated that the permeable form of NAADP (NAADP-AM) induces intracellular [Ca²⁺] increases in human sperm even in the absence of extracellular Ca²⁺. Using LysoTracker®, a fluorescent probe that selectively accumulates in acidic compartments, we identified two such stores in human sperm cells. Their acidic nature was further confirmed by the reduction in staining intensity observed upon inhibition of the endo-lysosomal proton pump with Bafilomycin, or after lysosomal bursting with glycyl-l-phenylalanine-2-naphthylamide. The selective fluorescent NAADP analog, Ned-19, stained the same subcellular regions as LysoTracker®, suggesting that these stores are the targets of NAADP action.