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Alberto Darszon

Publications and source records attributed to Alberto Darszon.

At least 19 recordsLinked to original sources
PubMed2006

Maitotoxin potently promotes Ca2+ influx in mouse spermatogenic cells and sperm, and induces the acrosome reaction.

Maitotoxin (MTX), a potent marine toxin, activates Ca2+ entry via nonselective cation channels in a wide variety of cells. The identity of the channels involved in MTX action remains unknown. In mammalian sperm, Ca2+ entry through store-operated channels regulates a number of physiological events including the acrosome reaction (AR). Here we report that MTX produced an increase in the intracellular concentration of Ca2+ ([Ca2+]i) in spermatogenic cells that depended on extracellular Ca2+. Ni2+ and SKF96365 diminished the MTX-activated Ca2+ uptake, at concentrations they inhibit store-operated channels, and in a similar manner as they inhibit the Ca2+ influx activated following depletion of intracellular stores by thapsigargin (Tpg). In addition, MTX significantly increased [Ca2+]i in single mature sperm and effectively induced the AR with a half-maximal concentration (ED50) of approximately 1.1 nM. Notably, SKF96365 similarly inhibited the MTX-induced increase in sperm [Ca2+]i and the AR triggered by the toxin, Tpg and zona pellucida. These results suggest that putative MTX-activated channels may be involved in the Ca2+ influx required for mouse sperm AR.

Acrosome↗
PubMed2005

Sodium and epithelial sodium channels participate in the regulation of the capacitation-associated hyperpolarization in mouse sperm.

In a process called capacitation, mammalian sperm gain the ability to fertilize after residing in the female tract. During capacitation the mouse sperm plasma membrane potential (E(m)) hyperpolarizes. However, the mechanisms that regulate sperm E(m) are not well understood. Here we show that sperm hyperpolarize when external Na(+) is replaced by N-methyl-glucamine. Readdition of external Na(+) restores a more depolarized E(m) by a process that is inhibited by amiloride or by its more potent derivative 5-(N-ethyl-N-isopropyl)-amiloride hydrochloride. These findings indicate that under resting conditions an electrogenic Na(+) transporter, possibly involving an amiloride sensitive Na(+) channel, may contribute to the sperm resting E(m). Consistent with this proposal, patch clamp recordings from spermatogenic cells reveal an amiloride-sensitive inward Na(+) current whose characteristics match those of the epithelial Na(+) channel (ENaC) family of epithelial Na(+) channels. Indeed, ENaC-alpha and -delta mRNAs were detected by reverse transcription-PCR in extracts of isolated elongated spermatids, and ENaC-alpha and -delta proteins were found on immunoblots of sperm membrane preparations. Immunostaining indicated localization of ENaC-alpha to the flagellar midpiece and of ENaC-delta to the acrosome. Incubations known to produce capacitation in vitro or induction of capacitation by cell-permeant cAMP analogs decreased the depolarizing response to the addition of external Na(+). These results suggest that increases in cAMP content occurring during capacitation may inhibit ENaCs to produce a required hyperpolarization of the sperm membrane.

Amiloride↗
PubMed2005

KATP channels in mouse spermatogenic cells and sperm, and their role in capacitation.

Mammalian sperm must undergo a series of physiological changes after leaving the testis to become competent for fertilization. These changes, collectively known as capacitation, occur in the female reproductive tract where the sperm plasma membrane is modified in terms of its components and ionic permeability. Among other events, mouse sperm capacitation leads to an increase in the intracellular Ca(2+) and pH as well as to a hyperpolarization of the membrane potential. It is well known that ion channels play a crucial role in these events, though the molecular identity of the particular channels involved in capacitation is poorly defined. In the present work, we report the identification and potential functional role of K(ATP) channels in mouse spermatogenic cells and sperm. By using whole-cell patch clamp recordings in mouse spermatogenic cells, we found K(+) inwardly rectifying (K(ir)) currents that are sensitive to Ba(2+), glucose and the sulfonylureas (tolbutamide and glibenclamide) that block K(ATP) channels. The presence of these channels was confirmed using inhibitors of the ATP synthesis and K(ATP) channel activators. Furthermore, RT-PCR assays allowed us to detect transcripts for the K(ATP) subunits SUR1, SUR2, K(ir)6.1 and K(ir)6.2 in total RNA from elongated spermatids. In addition, immunoconfocal microscopy revealed the presence of these K(ATP) subunits in mouse spermatogenic cells and sperm. Notably, incubation of sperm with tolbutamide during capacitation abolished hyperpolarization and significantly decreased the percentage of AR in a dose-dependent fashion. Together, our results provide evidence for the presence of K(ATP) channels in mouse spermatogenic cells and sperm and disclose the contribution of these channels to the capacitation-associated hyperpolarization.

ATP-Binding Cassette Transporters↗
PubMed2005

Cholesterol-depleting compounds modulate K+-currents in Drosophila Kenyon cells.

Sterol-enriched lipid rafts have been involved in Drosophila membrane signalling such as Hedgehog targeting and glutamate receptor ligand-affinity regulation. Here, we show that the voltage-dependent K(+) currents expressed by the intrinsic neurons of the Mushroom bodies are upward-modulated by compounds that remove sterols from the plasma membrane. Modulation seems to rely on a fast-exchanging sterol-pool, which more strongly affects the slowly inactivating current. Our results provide the first evidence that sterols influence the operation of voltage-gated ion channels in Drosophila neurons and strengthen the importance of lipid rafts in this biological model.

Animals↗
PubMed2005

A soluble adenylyl cyclase from sea urchin spermatozoa.

A previously identified, calmodulin-binding, sea urchin sperm flagellar adenylyl cyclase (AC) was cloned and sequenced and found to be a homologue of mammalian sperm soluble adenylyl cyclase (sAC). Compared to the mammalian sAC, the sea urchin sAC (susAC) has several long amino acid insertions, some of which contain protein kinase A phosphorylation sites. The enzymatic activity of susAC shows a steep pH dependency curve, the specific activity doubling when the pH is increased from 7.0 to 7.5. This suggests that like sperm dynein ATPase, the susAC is probably activated by increases in intracellular pH occurring upon spawning into seawater and also when sperm respond to contact with the egg jelly layer. The susAC is strongly activated by manganese, but has low activity in magnesium. Gene database searches identified sAC homologues in species known to have cyclic AMP-dependent sperm motility. This implies (as shown in mouse) that susAC has a role in sperm motility, most probably through axonemal protein phosphorylation or ion channel regulation.

Adenylyl Cyclases↗
PubMed2005

Real-time analysis of the role of Ca(2+) in flagellar movement and motility in single sea urchin sperm.

Eggs of many marine and mammalian species attract sperm by releasing chemoattractants that modify the bending properties of flagella to redirect sperm paths toward the egg. This process, called chemotaxis, is dependent on extracellular Ca(2+). We used stroboscopic fluorescence imaging to measure intracellular Ca(2+) concentration ([Ca(2+)]i) in the flagella of swimming sea urchin sperm. Uncaging of cyclic GMP induced Ca(2+) entry via at least two distinct pathways, and we identified a nimodipine-sensitive pathway, compartmentalized in the flagella, as a key regulator of flagellar bending and directed motility changes. We found that, contrary to current models, the degree of flagellar bending does not vary in proportion to the overall [Ca(2+)]i. Instead we propose a new model whereby flagella bending is increased by Ca(2+) flux through the nimodipine-sensitive pathway, and is unaffected by [Ca(2+)]i increases through alternative pathways.

Animals↗
PubMed2005

Shal and shaker differential contribution to the K+ currents in the Drosophila mushroom body neurons.

Shaker, a voltage-dependent K+ channel, is enriched in the mushroom bodies (MBs), the locus of olfactory learning in Drosophila. Mutations in the shaker locus are known to alter excitability, neurotransmitter release, synaptic plasticity, and olfactory learning. However, a direct link of Shaker channels to MB intrinsic neuron (MBN) physiology has not been documented. We found that transcripts for shab, shaw, shaker, and shal, among which only Shaker and Shal have been reported to code for A-type currents, are present in the MBs. The electrophysiological data showed that the absence of functional Shaker channels modifies the distribution of half-inactivation voltages (V(i1/2)) in the MBNs, indicating a segregation of Shaker channels to only a subset (approximately 28%) of their somata. In harmony with this notion, we found that approximately one-fifth of MBNs lacking functional Shaker channels displayed dramatically slowed-down outward current inactivation times and reduced peak-current amplitudes. Furthermore, whereas all MBNs were sensitive to 4-aminopyridine, a nonspecific A-type current blocker, a subset of neurons (approximately 24%) displayed little sensitivity to a Shal-specific toxin. This subset of neurons displaying toxin-insensitive outward currents had more depolarized V(i1/2) values attributable to Shaker channels. Our findings provide the first direct evidence that altered Shaker channel function disrupts MBN physiology in Drosophila. To our surprise, the experimental data also indicate that Shaker channels segregate to a minor fraction of MB neuronal somata (20-30%), and that Shal channels contribute the somatic A-type current in the majority of MBNs.

4-Aminopyridine↗
PubMed2005

Calcium channels and Ca2+ fluctuations in sperm physiology.

Generating new life in animals by sexual reproduction depends on adequate communication between mature and competent male and female gametes. Ion channels are instrumental in the dialogue between sperm, its environment, and the egg. The ability of sperm to swim to the egg and fertilize it is modulated by ion permeability changes induced by environmental cues and components of the egg outer layer. Ca(2+) is probably the key messenger in this information exchange. It is therefore not surprising that different Ca(2+)-permeable channels are distinctly localized in these tiny specialized cells. New approaches to measure sperm currents, intracellular Ca(2+), membrane potential, and intracellular pH with fluorescent probes, patch-clamp recordings, sequence information, and heterologous expression are revealing how sperm channels participate in fertilization. Certain sperm ion channels are turning out to be unique, making them attractive targets for contraception and for the discovery of novel signaling complexes.

Animals↗
PubMed2005

Sperm-activating peptide induces asymmetric flagellar bending in sea urchin sperm.

Speract, a sperm-activating peptide (SAP) from sea urchin eggs, induces various sperm responses including a transient increase in the intracellular Ca2+ concentration. However, it has not been clarified how speract modulates sperm motility and whether it functions as a chemoattractant. To confirm the effect of speract on sperm motility, we observed the flagellar bending response to speract in sperm of Hemicentrotus pulcherrimus, in experiments using caged speract and a lighting system for a microscope newly developed with a power LED. We found that speract induces increases in curvature of swimming paths and changes flagellar bending shape to asymmetric. These facts show that speract directly regulates flagellar motility, and suggest that speract-induced increases in intracellular Ca2+ concentration play an actual role in regulation of the flagellar movement.

Animals↗
PubMed2004

A sea urchin egg jelly peptide induces a cGMP-mediated decrease in sperm intracellular Ca(2+) before its increase.

Speract, a sperm-activating peptide (SAP) from sea urchin eggs, increases the intracellular concentration of Ca(2+) ([Ca(2+)]i) and modulates sperm motility. We measured the initial sperm response to speract using its caged analog and observed, for the first time, a small but significant decrease in sperm [Ca(2+)]i before the increase. Both directions of the [Ca(2+)]i change were completely blocked in high K(+) seawater. Using membrane-permeant caged cyclic nucleotides (cNMP), only cGMP induced the decrease in [Ca(2+)]i although both cGMP and cAMP increased the [Ca(2+)]i. The decrease in the [Ca(2+)]i induced by cGMP was more notable following a second photolytic event, once [Ca(2+)]i had been elevated by an initial flash. This pattern of [Ca(2+)]i change was confirmed in individual sperm. These results together with pharmacological evidence suggest that the initial [Ca(2+)]i decrease is due to a Na(+)/Ca(2+) exchanger activity, stimulated by hyperpolarization mediated by K(+) efflux through cGMP-regulated K(+) channels.

1-Methyl-3-isobutylxanthine↗
PubMed2004

Ion channel activity of membrane vesicles released from sea urchin sperm during the acrosome reaction.

The sperm acrosome reaction (AR) involves ion channel activation. In sea urchin sperm, the AR requires Ca2+ and Na+ influx and K+ and H+ efflux. During the AR, the plasma membrane fuses with the acrosomal vesicle membrane forming hybrid membrane vesicles that are released from sperm into the medium. This paper reports the isolation and preliminary characterization of these acrosome reaction vesicles (ARVs), using synaptosome-associated protein of 25 kDa (SNAP-25) as a marker. Isolated ARVs have a unique protein composition. The exocytosis regulatory proteins vesicle-associated membrane protein and SNAP-25 are inside ARVs, as judged by protease protection experiments, and membrane associated based on Triton X-114 partitioning. ARVs fused with planar bilayers display three main types of single channel activity. The most frequently recorded channel is cationic, weakly voltage dependent and has a low open probability that increases with negative potentials. This channel is activated by cAMP, blocked by Ba2+, and has a PK+/PNa+ selectivity of 4.5. ARVs represent a novel membrane preparation suitable to deepen our understanding of ion channel activity in the AR and during fertilization.

Acrosome Reaction↗
PubMed2004

Expression and differential cell distribution of low-threshold Ca(2+) channels in mammalian male germ cells and sperm.

Numerous sperm functions including the acrosome reaction (AR) are associated with Ca(2+) influx through voltage-gated Ca(2+) (Ca(V)) channels. Although the electrophysiological characterization of Ca(2+) currents in mature sperm has proven difficult, functional studies have revealed the presence of low-threshold (Ca(V)3) channels in spermatogenic cells. However, the molecular identity of these proteins remains undefined. Here, we identified by reverse transcription polymerase chain reaction the expression of Ca(V)3.3 mRNA in mouse male germ cells, an isoform not previously described in these cells. Immunoconfocal microscopy revealed the presence of the three Ca(V)3 channel isoforms in mouse spermatogenic cells. In mature mouse sperm only Ca(V)3.1 and Ca(V)3.2 were detected in the head, suggesting its participation in the AR. Ca(V)3.1 and Ca(V)3.3 were found in the principal and the midpiece of the flagella. All Ca(V)3 channels are also present in human sperm, but only to a minor extent in the head. These findings were corroborated by immunogold transmission electron microscopy. Tail localization of Ca(V)3 channels suggested they may participate in motility, however, mibefradil and gossypol concentrations that inhibit Ca(V)3 channels did not significantly affect human sperm motility. Only higher mibefradil doses that can block high-threshold (HVA) Ca(V) channels caused small but significant motility alterations. Antibodies to HVA channels detected Ca(V)1.3 and Ca(V)2.3 in human sperm flagella.

Animals↗
PubMed2003

ZD7288 inhibits low-threshold Ca(2+) channel activity and regulates sperm function.

In this study, ZD7288, a blocker of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, has been found to inhibit the mouse sperm acrosome reaction (AR). HCN channels have not yet been either recorded or implicated in mouse sperm AR, but low-threshold (T-type) Ca(2+) channels have. Interestingly, ZD7288 blocked native T-type Ca(2+) currents in mouse spermatogenic cells with an IC(50) of about 100 microM. This blockade was more effective at voltages producing low levels of inactivation, suggesting a differential affinity of ZD7288 for different channel conformations. Furthermore, ZD7288 inhibited all cloned T-type but not high-threshold N-type channels heterologously expressed in HEK-293 cells. Our results further support the role of T-type Ca(2+) channels in the mouse sperm AR.

Acrosome Reaction↗
PubMed2003

Transient receptor potential (TRPC) channels in human sperm: expression, cellular localization and involvement in the regulation of flagellar motility.

Capacitative Ca(2+) entry is a process whereby the activation of Ca(2+) influx through the plasma membrane is triggered by depletion of intracellular Ca(2+) stores. Some transient receptor potential (TRPC) proteins have been proposed as candidates for capacitative Ca(2+) channels. Recent evidence indicates that capacitative Ca(2+) entry participates in the sperm acrosome reaction (AR), an exocytotic process necessary for fertilization. In addition, several TRPCs have been detected heterogeneously distributed in mouse sperm, suggesting that they may participate in other functions such as motility. Using reverse transcription-polymerase chain reaction (RT-PCR) analysis, RNA messengers for TRPC1, 3, 6 and 7 were found in human spermatogenic cells. Confocal indirect immunofluorescence revealed the presence of TRPC1, 3, 4 and 6 differentially localized in the human sperm, and immunogold transmission electron microscopy indicated that TRPC epitopes are mostly associated to the surface of the cells. Because all of them were detected in the flagellum, TRPC channel antagonists were tested in sperm motility using a computer-assisted assay. Our results provide what is to our knowledge the first evidence that these channels may influence human sperm motility.

Calcium Channels↗
PubMed2003

Speract induces calcium oscillations in the sperm tail.

Sea urchin sperm motility is modulated by sperm-activating peptides. One such peptide, speract, induces changes in intracellular free calcium concentration ([Ca2+]i). High resolution imaging of single sperm reveals that speract-induced changes in [Ca2+]i have a complex spatiotemporal structure. [Ca2+]i increases arise in the tail as periodic oscillations; [Ca2+]i increases in the sperm head lag those in the tail and appear to result from the summation of the tail signal transduction events. The period depends on speract concentration. Infrequent spontaneous [Ca2+]i transients were also seen in the tail of unstimulated sperm, again with the head lagging the tail. Speract-induced fluctuations were sensitive to membrane potential and calcium channel blockers, and were potentiated by niflumic acid, an anion channel blocker. 3-isobutyl-1-methylxanthine, which potentiates the cGMP/cAMP-signaling pathways, abolished the [Ca2+]i fluctuations in the tail, leading to a very delayed and sustained [Ca2+]i increase in the head. These data point to a model in which a messenger generated periodically in the tail diffuses to the head. Sperm are highly polarized cells. Our results indicate that a clear understanding of the link between [Ca2+]i and sperm motility will only be gained by analysis of [Ca2+]i signals at the level of the single sperm.

1-Methyl-3-isobutylxanthine↗
PubMed2003

Scorpion toxins that block T-type Ca2+ channels in spermatogenic cells inhibit the sperm acrosome reaction.

The acrosome reaction (AR) is a Ca(2+)-dependent event required for sperm to fertilize the egg. The activation of T-type voltage-gated Ca(2+) channels plays a key role in the induction of this process. This report describes the actions of two toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively) on sperm Ca(2+) channels. Both toxins decrease T-type Ca(2+) channel activity in mouse spermatogenic cells and inhibit the AR in mature sperm. Saturating concentrations of the toxins inhibited at most approximately 70% of the whole-cell Ca(2+) current, suggesting the presence of a toxin-resistant component. In addition, both toxins inhibited approximately 60% of the AR, which is consistent with the participation of T-type Ca(2+) channels in the sperm AR.

Acrosome Reaction↗
PubMed2003

Intracellular sodium changes during the speract response and the acrosome reaction in sea urchin sperm.

The sperm-activating peptide speract and fucose-sulphate glycoconjugate (FSG) are sea urchin egg-envelope components that modulate sperm ion permeability. They influence motility and induce acrosomal reaction (AR), respectively. A fluorescent Na(+)-sensitive dye (Na(+)-binding benzofuran isophthalate, SBFI) was used to determine how these egg envelope components influence sperm Na(+) permeability. [Ca(2+)](i) and pH(i) were also measured to correlate their changes in response to speract and FSG with those observed in [Na(+)](i). SBFI determinations indicate that the resting [Na(+)](i) is 20 +/- 8 mM in sea urchin sperm. Saturating levels of speract increased [Na(+)](i) by approximately 15 mM, while similar levels of FSG caused a further elevation of approximately 30 mM. The kinetics of the [Na(+)](i), [Ca(2+)](i) and pH(i) changes induced by saturating levels of speract were faster than those induced by FSG. Both egg ligands appeared to activate more than one Na(+) transport system. Nifedipine, Ni(2+) and TEA(+) inhibited the ionic changes and the AR induced by FSG but, importantly, did not alter those caused by speract. Thus, there are differences in some of the ionic transport mechanisms that operate in the speract and FSG responses. ZD2788, a blocker of hyperpolarization and cyclic-nucleotide-gated (HCN) channels such as SpHCN present in sea urchin sperm, did not decrease the speract-induced [Na(+)](i) increase, but slowed its kinetics. Therefore, SpHCN does not play a major role in the uptake of Na(+) triggered by this decapeptide. KB-R7943, an inhibitor of Na(+)/Ca(2+) exchangers, decreased the resting [Na(+)](i) and did not change significantly the speract-induced [Ca(2+)](i) increase, but slowed its recovery.

Acrosome Reaction↗