Phosphoinositide-dependent pathways in mouse sperm are regulated by egg ZP3 and drive the acrosome reaction

Sperm of many animals must complete an exocytotic event, the acrosome reaction, in order to fuse with eggs. In mammals, acrosome reactions are triggered during sperm contact with the egg extracellular matrix, or zona pellucida, by the matrix glycoprotein ZP3. Here, we show that ZP3 stimulates production of phosphatidylinositol-(3,4,5)-triphosphate in sperm membranes. Phosphatidylinositol-3-kinase antagonists that prevent acrosome reactions and fertilization in vitro, while generation of this phosphoinositide in the absence of ZP3 triggered acrosome reactions. Downstream effectors of phosphatidylinositol-(3,4,5)-triphosphate in sperm include the protein kinases, Akt and PKCzeta. These studies outline a signal transduction pathway that plays an essential role in the early events of mammalian fertilization.     

Trp2 regulates entry of Ca2+ into mouse sperm triggered by egg ZP3

In many cells, receptor activation initiates sustained Ca2+ entry which is critical in signal transduction. Mammalian transient receptor potential (Trp) proteins, which are homologous to the Drosophila photoreceptor-cell Trp protein, have emerged as candidate subunits of the ion channels that mediate this influx. As a consequence of overexpression, these proteins produce cation currents that open either after depletion of internal Ca2+ stores or through receptor activation. However, determining the role of endogenous Trp proteins in signal transduction is complicated by the absence of selective antagonists. Here we examine Trp function during sperm-egg interaction. The sperm acrosome reaction is a Ca2+-dependent secretory event that must be completed before fertilization. In mammals, exocytosis is triggered during gamete contact by ZP3, a glycoprotein constituent of the egg's extracellular matrix, or zona pellucida (ZP). ZP3 activates trimeric G proteins and phospholipase C and causes a transient Ca2+ influx into sperm through T-type Ca2+ channels. These early responses promote a second Ca2+-entry pathway, thereby producing sustained increases in intracellular Ca2+ concentration ([Ca2+]i) that drive acrosome reactions. Our results show that Trp2 is essential for the activation of sustained Ca2+ influx into sperm by ZP3.     

Evidence that aggregation of mouse sperm receptors by ZP3 triggers the acrosome reaction

In the mouse, considerable evidence indicates that initial sperm binding to the zona pellucida (ZP) is mediated by ZP3. In addition, this same glycoprotein is also responsible for inducing the acrosome reaction (AR). Whereas the O-linked oligosaccharides of ZP3 appear to mediate sperm-ZP binding, the portion of ZP3 bearing AR activity has not been defined. To try to understand the bifunctional role of ZP3 (binding and AR inducing activities), we have examined the hypothesis that ZP3 aggregates sperm receptor molecules. By analogy with findings in a variety of other extracellular signal transducing systems, including receptors for growth factors and insulin, this aggregation event could initiate the cascade resulting in the AR. To test this hypothesis, we have generated monospecific polyclonal antibodies against ZP2 and against ZP3, and examined the effects of these probes on capacitated sperm incubated in the absence or presence of various ZP protein preparations. For some experiments, we have used proteolytic fragments of ZP3, a preparation known to retain specific binding, but not AR-inducing, activity. We show here that capacitated mouse sperm, incubated with ZP glycopeptides, displayed ARs when incubated subsequently with anti-ZP3 IgG; ARs did not occur when parallel sperm samples were incubated with anti-ZP2 IgG or with anti-ZP3 Fab fragments. When capacitated sperm were treated successively, with (a) ZP3 glycopeptides, (b) anti-ZP3 Fab fragments, and (c) goat anti-rabbit IgG, ARs occurred in the majority of sperm. An alternative approach to examine this hypothesis used ZP proteins obtained from tubal eggs treated previously with bioactive phorbol diester (12-O-tetradecanoyl phorbol-13-acetate [TPA]). This preparation arrests capacitated sperm in an intermediate state of the AR. We demonstrate here that these sperm can be induced to undergo a complete AR by subsequent treatment with anti-ZP3 IgG. Together, these findings are consistent with the hypothesis under examination, and suggest that the aggregation of sperm molecules recognized by ZP3 glycopeptides or by TPA-treated ZP is sufficient to trigger the events that occur during acrosomal exocytosis.     

Recombinant mouse ZP3 inhibits sperm binding and induces the acrosome reaction.

Mammalian fertilization involves interactions of sperm surface receptors with ligands of the zona pellucida, an extracellular matrix surrounding the ovulated egg. In mouse, the zona is composed of three glycoproteins. One of them, ZP3, participates in primary sperm binding and in the subsequent triggering of the sperm's acrosome reaction. Considerable evidence suggests that carbohydrate determinants of ZP3 are responsible for binding to sperm and may be important for acrosomal exocytosis. A full-length cDNA encoding mouse ZP3 was assembled and cloned into expression vectors that contained either a cytomegalovirus (CMV) or a vaccinia (P11) promoter. Mouse L-929 cells were stably transformed with the pZP3-CMV constructs, and green monkey CV-1 cells were infected with a recombinant vaccinia virus containing ZP3. rZP3 was affinity purified from culture media and detected on Western blots as a single 60- to 70-kDa band, which differed in molecular weight from native ZP3 (mean, 83 kDa). Nevertheless, rZP3 is biologically active. rZP3 decreases sperm-zona binding with a potency equivalent to that of native zona pellucida and, like native ZP3, rZP3 triggers acrosomal exocytosis in capacitated mouse sperm. Thus, rZP3 isolated from both rodent and primate cells appears to contain those carbohydrate and protein structures necessary for ZP3's dual role in fertilization.     

Ca(2+) entry activated by S-nitrosylation. Relationship to store-operated ca(2+) entry

The coupling between Ca(2+) pools and store-operated Ca(2+) entry channels (SOCs) remains an unresolved question. Recently, we revealed that Ca(2+) entry could be activated in response to S-nitrosylation and that this process was stimulated by Ca(2+) pool emptying (Favre, C. J., Ufret-Vincenty, C. A., Stone, M. R., Ma, H-T. , and Gill, D. L. (1998) J. Biol. Chem. 273, 30855-30858). In DDT(1)MF-2 smooth muscle cells and DC-3F fibroblasts, Ca(2+) entry activated by the lipophilic NO donor, GEA3162 (5-amino-3-(3, 4-dichlorophenyl)1,2,3,4-oxatriazolium), or the alkylator, N-ethylmaleimide, was observed to be strongly activated by transient external Ca(2+) removal, closely resembling activation of SOC activity in the same cells. The nonadditivity of SOC and NO donor-activated Ca(2+) entry suggested a single entry mechanism. Calyculin A-induced reorganization of the actin cytoskeleton prevented SOC but had no effect on GEA3162-induced Ca(2+) entry. However, a single entry mechanism could account for both SOC and NO donor-activated entry if the latter reflected direct modification of the entry channel by S-nitrosylation, bypassing the normal coupling process between channels and pools. Small differences between SOC and GEA3162-activated Ba(2+) entry and sensitivity to blockade by La(3+) were observed, and in HEK293 cells SOC activity was observed without a response to thiol modification. It is concluded that in some cells, S-nitrosylation modifies an entry mechanism closely related to SOC and/or part of the regulatory machinery for SOC-mediated Ca(2+) entry.     

Na(+) entry via store-operated channels modulates Ca(2+) signaling in arterial myocytes

In manynonexcitable cells, hormones and neurotransmitters activateNa⁺ influx and mobilizeCa²⁺ from intracellular stores.The stores are replenished by Ca²⁺influx via "store-operated"Ca²⁺ channels (SOC). The mainroutes of Na⁺ entry in these cellsare unresolved, and no role forNa⁺ in signaling has beenrecognized. We demonstrate that the SOC are a majorNa⁺ entry route in arterialmyocytes. Unloading of the Ca²⁺stores with cyclopiazonic acid (a sarcoplasmic reticulumCa²⁺ pump inhibitor) and caffeineinduces a large externalNa⁺-dependent rise in thecytosolic Na⁺ concentration. Onecomponent of this rise in cytosolicNa⁺ concentration is likely due toNa⁺/Ca²⁺exchange; it depends on elevation of cytosolicCa²⁺ and is insensitive to 10 mMMg²⁺ and 10 µMLa³⁺. Another component isinhibited by Mg²⁺ andLa³⁺, blockers of SOC; thiscomponent persists in cells preloaded with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid to buffer Ca²⁺ transients andpreventNa⁺/Ca²⁺exchange-mediated Na⁺ entry. ThisNa⁺ entry apparently is mediatedby SOC. The Na⁺ entry influencesNa⁺ pump activity andNa⁺/Ca²⁺exchange and has unexpectedly large effects on cell-wideCa²⁺ signaling. The SOC pathwaymay be a general mechanism by which Na⁺ participates in signaling inmany types of cells.

     

Relationship between the M42 antigen of mouse sperm and the acrosome reaction induced by ZP3

A murine monoclonal antibody, M42 mAb, directed against 200/220 Kd protein of mouse sperm, has been employed to study the molecular events of gamete interaction. We have reported previously that M42 mAb blocks mouse fertilization in a zona-dependent manner; the reagent specifically inhibits physiologically induced (zonae), but not pharmacologically induced (A23187), acrosome reactions in mouse sperm. Using solubilized mouse zonae pellucidae and purified ZP3, we demonstrate that M42 mAb inhibits acrosome reactions (ARs) induced by ZP3 to the same extent as those induced by total zonae. We have also studied AR inhibition using the fluorescent antibiotic chlortetracycline (CTC), which permits visualization of three different acrosomal patterns during the AR. In the presence of M42 IgG, greater than 70% of capacitated sperm treated with zonae are arrested in the acrosome-intact state (B-pattern), in contrast to the majority of sperm (60-70%) in the absence of M42 IgG, which progress through the intermediate phase (S-pattern) to the fully acrosome-reacted (AR-pattern) state. Incubation of sperm with zona proteins modified by incubating eggs with phorbol esters arrests sperm in the S-pattern (Y. Endo, R.M. Schultz, and G.S. Kopf, 1987, Dev. Biol. 119, 199-209). We show that once sperm have reached such a state, M42 mAb no longer exerts an inhibitory effect. The addition of unmodified ZP to S-pattern sperm permits the completion of the acrosome reaction. These results indicate that M42 mAb blocks an early step in the AR cascade and that M42 mAb is unable to prevent subsequent events of this cascade once it has been initiated.     

Arachidonic acid in astrocytes blocks Ca(2+) oscillations by inhibiting store-operated Ca(2+) entry,and causes delayed Ca(2+) influx

ATP-elicited oscillations of the concentration of free intracellular Ca(2+) ([Ca(2+)](i)) in rat brain astrocytes were abolished by simultaneous arachidonic acid (AA) addition, whereas the tetraenoic analogue 5,8,11,14-eicosatetraynoic acid (ETYA) was ineffective. Inhibition of oscillations is due to suppression by AA of intracellular Ca(2+) store refilling. Short-term application of AA, but not ETYA, blocked Ca(2+) influx, which was evoked by depletion of stores with cyclopiazonic acid (CPA) or thapsigargin (Tg). Addition of AA after ATP blocked ongoing [Ca(2+)](i) oscillations. Prolonged AA application without or with agonist could evoke a delayed [Ca(2+)](i) increase. This AA-induced [Ca(2+)](i) rise developed slowly, reached a plateau after 5 min, could be reversed by addition of bovine serum albumin (BSA), that scavenges AA, and was blocked by 1 microM Gd(3+), indicative for the influx of extracellular Ca(2+). Specificity for AA as active agent was demonstrated by ineffectiveness of C16:0, C18:0, C20:0, C18:2, and ETYA. Moreover, the action of AA was not affected by inhibitors of oxidative metabolism of AA (ibuprofen, MK886, SKF525A). Thus, AA exerted a dual effect on astrocytic [Ca(2+)](i), firstly, a rapid reduction of capacitative Ca(2+) entry thereby suppressing [Ca(2+)](i) oscillations, and secondly inducing a delayed activation of Ca(2+) entry, also sensitive to low Gd(3+) concentration.     

Calmodulin antagonists inhibit T-type Ca(2+) currents in mouse spermatogenic cells and the zona pellucida-induced sperm acrosome reaction.

The sperm acrosome reaction (AR) is a regulated exocytotic process required for gamete fusion. It depends on an increase in [Ca(2+)](i) mediated by Ca(2+) channels. Although calmodulin (CaM) has been reported to regulate several events during the AR, it is not known whether it modulates sperm Ca(2+) channels. In the present study we analyzed the effects of CaM antagonists W7 and trifluoroperazine on voltage-dependent T-type Ca(2+) currents in mouse spermatogenic cells and on the zona pellucida-induced AR in sperm. We found that these CaM antagonists decreased T-currents in a concentration-dependent manner with IC(50) values of approximately 10 and approximately 12 microM, respectively. W7 altered the channels' voltage dependence of activation and slowed both activation and inactivation kinetics. It also induced inactivation at voltages at which T-channels are not activated, suggesting a promotion of inactivation from the closed state. Consistent with this, W7 inhibited the ZP-induced [Ca(2+)](i) transients in capacitated sperm. Likewise, W7 and TFP inhibited the AR with an IC(50) of approximately 10 microM. In contrast, inhibitors of CaM-dependent kinase II and protein kinase A, as well as a CaM-activated phosphatase, had no effect either on T-currents in spermatogenic cells or on the sperm AR. Together these results suggest a functional interaction between CaM and the sperm T-type Ca(2+) channel. They are also consistent with the involvement of T-channels in the AR.     

A sustained increase in intracellular Ca(2+) is required for the acrosome reaction in sea urchin sperm.

The acrosome reaction (AR), necessary for fertilization in many species, requires an increase in intracellular Ca(2+) ([Ca(2+)](i)). In sea urchin sperm, the AR is triggered by an egg-jelly factor: the associated [Ca(2+)](i) elevation lasts minutes and involves two Ca(2+) permeable channels. Both the opening of the second channel and the onset of the AR occur approximately 5 s after treatment with egg factor, suggesting that these events are linked. In agreement, removal of Ca(2+) from sea water or addition of Ca(2+) channel blockers at the time when opening of the second channel is first detected inhibits AR and causes a "rapid" (t(1/2) = 3--15 s) decrease in [Ca(2+)](i) and partial inhibition of the intracellular pH change associated with the AR. Simultaneous addition of NH(4)Cl and either EGTA, Co(2+), or Ni(2+) 5 s after egg factor prevents the partial inhibition of the evoked pH(i) change observed but does not reverse AR inhibition. Therefore, the sustained increase in [Ca(2+)](i) caused by the second Ca(2+) channel is needed for the sperm AR. Experiments with agents that induce capacitative Ca(2+) uptake (thapsigargin and cyclopiazonic acid) suggest that the second channel opened during the AR could be a store-operated Ca(2+) channel.     

A role for the human sperm glycine receptor/Cl(-) channel in the acrosome reaction initiated by recombinant ZP3.

Previously, we have demonstrated an essential role for the neuronal glycine receptor (GlyR) in the acrosome reaction (AR) of mouse and porcine sperm initiated by the egg zona pellucida (ZP). In the present study, we have demonstrated presence of the GlyR in human sperm by immunoprecipitation and Western blot analysis, investigated the potential of a recombinant human ZP3 (rhZP3) preparation as an alternative research tool to solubilized human ZP, and shown that the human sperm GlyR is essential to the human AR initiated by rhZP3. Additionally, we have been able to demonstrate that rhZP3 possesses biological activity, because it is able to rapidly stimulate the AR in capacitated human sperm and its action is blocked by the addition of pertussis toxin. Moreover, spectrofluorometric studies using fura-2-loaded human sperm have shown that rhZP3 triggers a peak-and-plateau rise in intracellular Ca(2+) levels similar to that seen with solubilized mammalian ZP. These results suggest that the actions of rhZP3 and solubilized ZP are elicited via the same signal transduction pathways. Furthermore, incubation of human sperm with an antibody directed against the alpha1 subunit of the human spinal cord GlyR or with 50 nM strychnine caused significant inhibition in the rhZP3-initated AR. Finally, studies using fura-2-loaded human sperm showed that 50 nM strychnine was also able to inhibit the Ca(2+) influx associated with addition of rhZP3. These results further support the view that rhZP3 and the ZP work through the same mechanisms, show that the GlyR is involved in rhZP3-initiated AR, and suggest that the GlyR may also play a role in the early signal transduction cascades associated with ZP-initiated AR in vivo.     

A transient rise in intracellular Ca2+ is a precursor reaction to the zona pellucida-induced acrosome reaction in mouse sperm and is blocked by the induced acrosome reaction inhibitor 3-quinuclidinyl benzilate.

The acrosome reaction induced by the zona pellucida in mouse sperm has been shown to proceed in two stages experimentally distinguishable by the fluorescent probe chlortetracycline. Entry into the first stage of sperm bound to isolated, structurally intact zonae pellucidae is blocked by the compound 3-quinuclidinyl benzilate. In this study, we show, utilizing the fluorescent Ca2+ indicator fluo-3, that the first stage of the zona-induced acrosome reaction is characterized by an increase in intracellular Ca2+, followed by a decrease as the acrosome reaction proceeds. This calcium transient is completely suppressed by 3-quinuclidinyl benzilate. We conclude that the Ca2+ transient is induced by the zona pellucida and is required for the zona-induced acrosome reaction. Blockage of this sperm intracellular Ca2+ transient provides a mechanism for the inhibitory action of 3-quinuclidinyl benzilate on the zona-induced acrosome reaction in mouse sperm.     

Heteromeric TRPV4-C1 channels contribute to store-operated Ca(2+) entry in vascular endothelial cells

There is controversy as to whether TRP channels participate in mediating store-operated current (I_SOC) and store-operated Ca²⁺ entry (SOCE). Our recent study has demonstrated that TRPC1 forms heteromeric channels with TRPV4 in vascular endothelial cells and that Ca²⁺ store depletion enhances the vesicle trafficking of heteromeric TRPV4-C1 channels, causing insertion of more channels into the plasma membrane in vascular endothelial cells. In the present study, we determined whether the enhanced TRPV4-C1 insertion to the plasma membrane could contribute to SOCE and I_SOC. We found that thapsigargin-induced SOCE was much lower in aortic endothelial cells derived from trpv4^−/− or trpc1^−/− knockout mice when compared to that of wild-type mice. In human umbilical vein endothelial cells (HUVECs), thapsigargin-induced SOCE was markedly reduced by knocking down the expression of TRPC1 and/or TRPV4 with respective siRNAs. Brefeldin A, a blocker of vesicular translocation, inhibited the SOCE. These results suggest that an enhanced vesicular trafficking of heteromeric TRPV4-C1 channels contributes to SOCE in vascular endothelial cells. Vascular tension studies suggest that such an enhanced trafficking of TRPV4-C1 channels may play a role in thapsigargin-induced vascular relaxation in rat small mesenteric arteries.     

Relation between phosphatidylserine exposure and store-operated Ca(2+) entry in stimulated cells

A significant increase in intracellular Ca(2+) is required to trigger the remodeling of the cell plasma membrane. Scott syndrome is an extremely rare inherited disorder of the transmembrane migration of phosphatidylserine toward the exoplasmic leaflet in blood cells. We have recently reported a reduced capacitative Ca(2+) entry in Scott cells [Martínez et al. (1999) Biochemistry 38, 10092-10098]. We have investigated here the links between defective phosphatidylserine exposure and Ca(2+) signaling in Scott cells by focusing on the Ca(2+) entry following the emptying of intracellular stores. After depletion of caffeine- or thapsigargin-sensitive stores, Ca(2+) entry was lower in Scott compared to control lymphoblasts. However, the simultaneous depletion of both types of stores restored a normal Ca(2+) influx across the plasma membrane in Scott cells and phosphatidylserine externalization ability was improved concomitantly with capacitative Ca(2+) entry. These observations point to the essential role of capacitative Ca(2+) entry in the control of phosphatidylserine exposure of stimulated cells.     

KB-R7943 inhibits store-operated Ca(2+) entry in cultured neurons and astrocytes

We have studied cyclopiazonic acid (CPA)-sensitive store-operated Ca(2+) entry (SOCE) in cultured neurons and astrocytes and examined the effect of 2-[2-[4-(4-nitrobenzyloxy)phenyl]]isothiourea (KB-R7943), which is often used as a selective inhibitor of the Na(+)-Ca(2+) exchanger (NCX), on the SOCE. CPA increased transiently intracellular Ca(2+) concentration ([Ca(2+)](i)) followed by a sustained increase in [Ca(2+)](i) in neurons and astrocytes. The sustained increase in [Ca(2+)](i) depended on the presence of extracellular Ca(2+) and inhibited by SOCE inhibitors, but not by a Ca(2+) channel inhibitor. CPA also caused quenching of fura-2 fluorescence when the cells were incubated in Mn(2+)-containing medium. KB-R7943 at 10 microM inhibited significantly CPA-induced sustained increase in [Ca(2+)](i) in neurons and astrocytes. KB-R7943 also inhibited CPA-induced quenching of fura-2 fluorescence in the presence of extracellular Mn(2+). These results indicate that cultured neurons and astrocytes possess SOCE and that KB-R7943 inhibits not only NCX but also SOCE.     

Evidence for the activation of two different Ca2+ channels during the egg jelly-induced acrosome reaction of sea urchin sperm

The influx of Ca2+ and its subsequent intracellular increase are required for the acrosome reaction of sea urchin sperm to occur. Spermatozoa must undergo this reaction, which is triggered by the egg jelly, in order to fertilize the egg. Here, the egg jelly-induced Ca2+ influx mechanisms have been studied in sperm loaded with FURA-2 using Mn2+ under the assumption that this divalent ion is an indicator of Ca2+ influx through Ca2+ channels. Egg jelly induced the immediate entry of Ca2+ (mixing time 1 s), however; we found that the influx of Mn2+ increased after a lag time of 5 s. Nisol-dipine (a Ca2+ channel blocker) did not block the Mn2+ influx which was inhibited by 40 mM of external [K+], low Na+, and 5 mM of tetraethylammonium (a K+ channel blocker). These conditions also inhibited the alkalinization and the acrosome reaction. The inhibition of the Mn2+ influx could be overcome by increasing internal pH (pHi) with ammonium (10 mM). On the contrary the influx of Ca2+ during the first 5 s was not inhibited by any of the conditions indicated before, except by nisoldipine. These data could be explained by the activation of two different Ca2+ channels by egg jelly. The first one being a receptor-operator Ca2+ channel that opens when the receptor for egg jelly is occupied independently of the ionic conditions. The other one could be considered as a second messenger-operated Ca2+ channel that requires at least an increase in pHi to open.     

Relocalization of STIM1 for activation of store-operated Ca(2+) entry is determined by the depletion of subplasma membrane endoplasmic reticulum Ca(2+) store

STIM1 (stromal interacting molecule 1), an endoplasmic reticulum (ER) protein that controls store-operated Ca(2+) entry (SOCE), redistributes into punctae at the cell periphery after store depletion. This redistribution is suggested to have a causal role in activation of SOCE. However, whether peripheral STIM1 punctae that are involved in regulation of SOCE are determined by depletion of peripheral or more internal ER has not yet been demonstrated. Here we show that Ca(2+) depletion in subplasma membrane ER is sufficient for peripheral redistribution of STIM1 and activation of SOCE. 1 microM thapsigargin (Tg) induced substantial depletion of intracellular Ca(2+) stores and rapidly activated SOCE. In comparison, 1 nM Tg induced slower, about 60-70% less Ca(2+) depletion but similar SOCE. SOCE was confirmed by measuring I(SOC) in addition to Ca(2+), Mn(2+), and Ba(2+) entry. Importantly, 1 nM Tg caused redistribution of STIM1 only in the ER-plasma membrane junction, whereas 1 microM Tg caused a relatively global relocalization of STIM1 in the cell. During the time taken for STIM1 relocalization and SOCE activation, 1 nM Bodipy-fluorescein Tg primarily labeled the subplasma membrane region, whereas 1 microM Tg labeled the entire cell. The localization of Tg in the subplasma membrane region was associated with depletion of ER in this region and activation of SOCE. Together, these data suggest that peripheral STIM1 relocalization that is causal in regulation of SOCE is determined by the status of [Ca(2+)] in the ER in close proximity to the plasma membrane. Thus, the mechanism involved in regulation of SOCE is contained within the ER-plasma membrane junctional region.     

A nicotinic acetylcholine receptor is involved in the arosome reaction of human sperm initiated by recombinant human ZP3

One of the essential steps in mammalian fertilization is the acrosome reaction (AR), a modified exocytotic event in the sperm head that occurs upon contact with the glycoprotein matrix of the zona pellucida (ZP) surrounding the oocyte. Acetylcholine (ACh) at concentrations of 10-250 micro M and nicotine at 10-250 nM significantly initiate the AR of capacitated human sperm. Preincubation with three antagonists of the nicotinic acetylcholine receptor (nAChR), alpha-bungarotoxin (alpha-BTX, 100 nM), alpha-conotoxin IMI (alpha-CTX IMI, 250 nM and 25 nM), and methyllycaconitine (MLA, 100 nM and 10 nM), significantly blocked AR initiation by ACh. alpha-BTX is an anatagonist of several nAChRs, including the alpha7 nAChR, and alpha-CTX IMI and MLA are highly specific antagonists of alpha7 subunit-containing AChRs. The sperm nAChR plays a role in the AR initiated in vitro by a purified recombinant human ZP protein (rhZP3). Previously, rhZP3 was able to stimulate the AR by mechanisms similar to those seen with native ZP. Preincubation of human sperm with alpha-BTX (from 10 micro M to 100 nM), alpha-CTX IMI (250 and 100 nM), or MLA (100 nM and 10 nM) caused a significant inhibition in the rhZP3-initated AR. The inhibition of the ACh-initiated and rhZP3-initiated AR by these nAChR antagonists strongly suggests the involvement of an alpha7 subunit-containing nAChR in the AR initiated by both ligands. AR initiation by progesterone was not inhibited by MLA or alpha-BTX, suggesting that this particularnAChR is not involved in the AR initiated by that ligand. In vitro results show for the first time that ACh can initiate the human sperm AR and strongly suggest that a human sperm alpha7 subunit-containing nAChR plays a role in the rhZP3-initiated AR. This nAChR ligand-gated ion channel may be important to the signal transduction events of ZP-initiated AR in vivo.     

Molecular evolution and functional divergence of the Ca(2+) sensor protein in store-operated Ca(2+) entry: stromal interaction molecule

Receptor-mediated Ca(2+) signaling in many non-excitable cells initially induces Ca(2+) release from intracellular Ca(2+) stores, followed by Ca(2+) influx across the plasma membrane. Recent findings have suggested that stromal interaction molecules (STIMs) function as the Ca(2+) sensor to detect changes of Ca(2+) content in the intracellular Ca(2+) stores. Human STIMs and invertebrate STIM share several functionally important protein domains, but diverge significantly in the C-terminus. To better understand the evolutionary significance of STIM activity, phylogenetic analysis of the STIM protein family was conducted after extensive database searching. Results from phylogeny and sequence analysis revealed early adaptation of the C-terminal divergent domains in Urochordata, before the expansion of STIMs in Vertebrata. STIMs were subsequently subjected to one round of gene duplication as early as in the Euteleostomi lineage in vertebrates, with a second round of fish-specific gene duplication. After duplication, STIM-1 and STIM-2 molecules appeared to have undergone purifying selection indicating strong evolutionary constraints within each group. Furthermore, sequence analysis of the EF-hand Ca(2+) binding domain and the SAM domain, together with functional divergence studies, identified critical regions/residues likely underlying functional changes, and provided evidence for the hypothesis that STIM-1 and STIM-2 might have developed distinct functional properties after duplication.